Theriogenology xxx (2014) 1–10
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Effects of physiological and/or disease status on the response of postpartum dairy cows to synchronization of estrus using an intravaginal progesterone device Julie C. McNally a, Mark A. Crowe a, b, *, James F. Roche a, Marijke E. Beltman a a b
School of Veterinary Medicine, University College Dublin, Belfield, Dublin, Ireland Conway Institute for Biomolecular and Biomedical Research, University College Dublin, Belfield, Dublin, Ireland
a r t i c l e i n f o
a b s t r a c t
Article history: Received 26 May 2014 Received in revised form 13 August 2014 Accepted 13 August 2014
Progesterone treatments are used to increase submission rates in postpartum dairy cows; however, in many cases the protocol is used as a blanket therapy for all cows without regard for physiological or disease state. The objective of this study was to identify the physiological or disease classes of cows that respond well (or not) to synchronization of estrus via progesterone. Dairy cows (n ¼ 402) were monitored peri and postpartum to establish their physiological or disease status. Animals were classified as having negative energy balance, clinical lameness, uterine infection (UI), anovulatory anestrus, high somatic cell counts, and healthy (H). Blood samples were collected at five different time points and analyzed for metabolites. All animals received an 8-day controlled internal drug release protocol, which included GnRH at insertion and PGF2a the day before removal. Response to the protocol was determined by visual observation of estrus synchronization. Conception rate was determined by ultrasonography between Days 32 and 35 after artificial insemination. Animals without UI were 1.9 times more likely to respond and two times more likely to be confirmed pregnant than those with UI. There was no relationship between negative energy balance and clinical lameness in the visual estrous response, but both conditions were associated with reduced conception rates. Dairy cows in anovulatory anestrus responded successfully to the protocol in both estrous response and conception rates. High glutathione peroxidase concentrations had a positive effect on conception rates, whereas high non-esterified fatty acids and beta-hydroxybutyrate had a negative effect on the estrous response. In conclusion, disease and physiological states of dairy cows determined the response to progesterone-based synchronization. The more disease or physiological problems the cows had, the lower the estrous response and conception rates; cows with these problems were not ideal candidates for synchronization. Both anestrus and healthy dairy cows were good responders to progesterone-based synchronization. Ó 2014 Elsevier Inc. All rights reserved.
Keywords: Dairy cattle Estrous synchronization Physiological state Disease
1. Introduction Modern dairy production is based on high-yielding dairy cows producing over 9000 kg per lactation. One of
* Corresponding author. Tel.: þ353 1 716 6255; fax: þ353 (0)1 716 6255. E-mail address: [email protected] (M.A. Crowe). 0093-691X/$ – see front matter Ó 2014 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.theriogenology.2014.08.006
the key problem areas decreasing the efficiency and sustainability of milk production is infertility. In Irish dairy herds, cow fertility measured as calving rate to a single insemination declined at a rate of almost 1% per annum (55% to 44%) in the period from 1990 to 2001 [1]. Infertility has large economic impacts, and the importance of infertility for dairy farmers was emphasized by the results of a recent Delphi study [2] where infertility was one of the main concerns of farmers. Cows selected for high milk
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production may be more prone to an increased incidence of diseases such as endometritis, mastitis, milk fever, and lameness, as well as a higher risk of going into negative energy balance (NEB) postpartum, all of which have a negative effect on their reproductive status [3,4]. Apart from the reduced conception rates, some of the key causes of low reproductive efficiency in postpartum high-yielding dairy cows are as follows: (1) an increased incidence of clinical or subclinical endometritis, (2) a larger postpartum interval to first ovulation, (3) increased incidence of irregular cycles, (4) shorter duration and less intense expression of estrus synchronization, and (5) reduced conception rate per single insemination often followed by an increase in fetal loss [5]. The factors contributing to infertility problems in postpartum dairy cows are multifactorial and include genetics, nutrition, health, welfare, and poor reproductive management. Reproductive performance influences profitability of a dairy cow–calf operation. A high reproductive performance is especially important for seasonal dairy herds based on pasture where breeding and calving are restricted to a limited period of the year in order to match the feed requirements to the seasonal pattern of pasture growth [6]. The ability to synchronize onset of estrus, and hence the time of breeding and calving, offers potential economic and management benefits to dairy farmers. It increases the rate of genetic gain through more usage of artificial insemination (AI) with genetically superior proven sires with high-accuracy expected progeny differences, increases milk production through advancement of the mean calving date, and reduces the percentage of nonpregnant cows at the end of the breeding season [6]. The transition period of 3 weeks pre-calving until 3 weeks post-calving is associated with a peak incidence of production diseases that may disrupt animal fertility. Although traditionally regarded as the significant metabolic disorders of dairy cows (hypocalcemia, hypomagnesemia, and ketosis), the term production disease, which is influenced by high production and management of the transition cow, has been broadened to include conditions such as retained placenta, uterine discharge, milk fever, mastitis, and lameness [7,8]. The precise cause of impaired immune function in transition cows is unclear, although the peripartum decrease in energy, vitamin, and mineral intake, which can result in NEB and mobilization of body fat are contributing factors. The dramatic changes in progesterone and estrogen levels in late gestation also seem to play a role [9–11]. Lame cows have a lower overall pregnancy rate and 65% of the annual incidence of lameness occurs within the first month of lactation; therefore, lameness is one of the major reasons for culling in dairy herds [12]. Postpartum uterine disease is also a component of reproductive performance [13]. Butler and Smith [14] found that cows that lost less than 0.5 units of body condition score (BCS) during the first 5 weeks postpartum had higher conception rates at the first service than cows that lost more than 0.5 BCS. Clinical mastitis or high somatic cell count (SCC) in the postpartum period delays the resumption of ovarian activity considerably [15,16]. Our hypothesis was that the response to progesterone-based synchronization in dairy cows is
dependent on the physiological and disease status of the animal in the postpartum period. Both progestagens and prostaglandins are used routinely for estrous synchronization in cattle. Progesterone-based methods to synchronize estrus result in less variation in the timing of onset of estrus compared with prostaglandinbased methods [17], and they may be used to induce estrus in anestrous cows in the postpartum period. In the current study, an 8-day progesterone-based treatment, which included GnRH at the insertion and PGF2a the day before removal, was used. The objective of this experiment was to determine the response to progesterone-based synchronization in dairy cows in relation to their physiological and disease status with the aim to identify the physiological classes of cows that respond well (or do not respond well) to a synchronization protocol including progesterone. 2. Materials and methods 2.1. Study animals All experimental procedures involving animals were licensed in accordance with the Cruelty to Animals Act (Ireland 1876) and the European Community Directive 86/ 609/EC and were sanctioned by the University College Dublin Animal Research Ethics Committee. This study was conducted using multiparous (N ¼ 250) and primiparous cows (N ¼ 152) from five commercial spring-calving dairy herds in the Leinster region (Ireland) between November 2011 and July 2012. Cows of all parities (1 to 11) were represented; median lactation number was 2 and maximum was 11, with normal (no intervention, calving on own) and low degrees (minimal intervention by the farmer) of calving difficulty that calved between January and April 2012 were enrolled in the trial. All animals were housed indoors within a free-stall barn during their 60 days dry period before calving. After parturition, they remained indoors within a free-stall barn until turn out to pasture in spring (varied from early February to April across the farms) with the exception of one herd that remained indoors throughout the study. Breeds represented were Holstein (n ¼ 100), Holstein-Friesian (n ¼ 203), and Crossbreds (n ¼ 99). The distribution of breeds on individual farms ranged from primarily HolsteinFriesian to primarily crossbreds. 2.2. Experimental procedures All cows enrolled on the trial were evaluated at seven time points around calving. At each sample/evaluation time, locomotion score (LM), BCS, rectal temperature as a measure of overall health, and vaginal mucus score (MS) as a measurement of uterine health were recorded. In addition, blood samples for metabolic parameters were collected. Insulin-like growth factor-I (IGF-I), glutathione peroxidase (GPX; as an estimate of the selenium status) were analyzed at five time points, beta-hydroxybutyrate (BHB), nonesterified fatty acids (NEFAs), and urea were analyzed at three time points, calcium (Ca), magnesium (Mg), and phosphorus (P) were analyzed at one time point. A timeline of these seven time points is represented in Table 1.
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postpartum. Animals classified as NEB had a loss of 0.5 of a BCS from calving to Day 32 4 postpartum. Animals that had a locomotion score of 3 post-calving from Days 0 to 32 4 were classified as clinically lame [18]. The animals classified in the SSC 200,000 cells/mL were defined as those having an SCC 200,000 cells/mL 30 days before breeding. Animals classified as anovulatory anestrus (AA) had clean uterine horns with no evidence of inflammation or infection but had delayed ovulation (absence of detectable CL when ovaries were scanned up to Day 37 9 and at controlled internal drug release (CIDR) insertion). The animals that were in none of the above classifications were then categorized as healthy (H). All herds were tested for Johnes, bovine viral diarrhea, infectious bovine rhinotracheitis, leptospirosis, liver, and rumen flukes through a bulk milk sample. This sample was taken when all the cows on the trial were postpartum and tested using an ELISA bulk milk sampling kit. Individual milk recording for SCC took place by a commercial company four times during the trial period. Body temperature was recorded on Day 10 4 postpartum using a digital rectal thermometer.
Locomotion was scored as described by Spreacher et al. [16], where a score of 1 represented normal locomotion and 2 represented mild lameness; these animals showed an arched back while walking, but their gait remained normal. An animal with a locomotion score of 3 displayed an arched back when both standing and walking and an animal with a score of 4 showed an arched back while standing and walking and a gait that is one deliberate step at a time. Body condition was scored on a 5-point scale with increments of 0.25 (as described by Edmonson et al. [19]). Blood samples were taken at five time points (Days 7 7 pre-calving, 4 4, 18 4, 25 3, and 32 4 postpartum) by coccygeal venipunture. Blood samples were collected into three Vacutainer tubes (Becton, Dickinson and Company, Plymouth, UK), two containing lithium heparin for plasma collection and one containing a clot activator for serum collection. At each blood time point, a plasma and serum sample was stored at ambient temperature for a maximum of 6 hours, and then at 4 C for a further 18 hours; each sample was decanted after centrifugation for 20 minutes at 1600 g and stored at 20 C until subsequent analysis. A plasma sample was assayed for GPX within 24 hours of collection. Vaginal mucus was collected by means of a metricheck (Simcro Tech, Hamilton, New Zealand) device that scoops discharge from the anterior vagina. The mucus was assessed by one person for color, proportion, and volume of discharge, and a character score assigned as follows: (0) clear or translucent mucus; (1) mucus containing flecks of white or off-white pus; (2) less than 50 mL exudates containing less than 50% white or off-white mucopurulent material; and (3) more than 50 mL exudates containing purulent material, usually white or yellow, but occasionally sanguineous [20]. The vaginal mucus was also assessed by odor, and given a score 0 for normal odor or a score of 1 if a fetid odor was detected [21]. The metricheck device was cleaned and placed in suitable disinfectant between each cow sampled. The mucus scoring was carried out at three time points postpartum (Days 18 4, 23 5, and 32 4). The SCC of each animal was recorded monthly, and animals were assigned to a grade according to their SCC score. Grade 1 was animals with a score less than200,000; grade 2 animals had an SCC 200,000. Animals were assigned to a classification according to their disease or physiological state. Animals were assigned to the uterine infection (UI) classification when the metricheck score was 2 [21] on Days 23 5 and 32 4
2.3. Synchronization treatments and AI Estrous cycles of all animals were synchronized using a CIDR (containing 1.38 g progesterone, Pfizer Animal Health, Dublin, Ireland; now Zoetis) intravaginally for 8 days, with a 2-mL im injection of a GnRH (Receptal, MSD Animal Health, Ireland) at the time of CIDR insertion and a 2.5-mL im injection of PGF2a analogue (Estrumate, MSD Animal Health) 1 day before CIDR withdrawal. All cows were 37 days in milk (DIM) (mean ¼ 59; range 37–93 DIM) at the initiation of synchrony treatment, resulting in synchronized estrus or ovulation at 47 DIM (mean ¼ 69; range ¼ 47–103 DIM). Cows were inseminated by the AM/ PM rule (a cow observed in standing estrus in the morning was inseminated in the afternoon of the same day and a cow observed in standing estrus in the afternoon or evening was bred the following morning), following detection of estrus with the aid of tail paint. Detection of estrus was performed four times per 24-hour period for 20 minutes (i.e., 6-hour intervals). All inseminations were performed by experienced technicians from commercial AI companies or by the herd owners and farm staff licensed by the Department of Agriculture, Fisheries and Food (Ireland), to carry out AI.
Table 1 Timeline of key sample and data collection points relative to day of calving for dairy cows enrolled in the study. Time point
Days from calving
BCS
LM
1 2 3 4 5 6 7
60 to 40 21 to 0 0–7 7–14 14–21 21–28 28–35 w50
U U U
U
Mucus
Temperature
U
Serum
Plasma
U U
U U
U U U
U U U
Scan
U
U CIDR insertion
U U U
Abbreviations: BCS, body condition score; CIDR, controlled internal drug release; LM, locomotion score.
U U U
4
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2.4. Transrectal ultrasonography The reproductive tracts of all cows were examined by transrectal ultrasound on Days 25 3 and 32 4 postcalving and immediately before initiation of synchronization. One experienced operator using an Ibex Lite ultrasound scanner (Ibex Lite; E.I Medical Imaging, CO, USA) equipped with a 5-MHz transrectal transducer performed the examinations. Cows were assigned an ultrasound reproductive tract score describing the volume and echogenicity of fluid contained within the uterus [22]. All cows were scanned at 32 to 35 days after AI to confirm pregnancy; this was confirmed by palpation per rectum, visualization of a fluid-filled uterine horn, and the presence of a conceptus as positive indicators of pregnancy. The uterine, ovarian, and pregnancy findings were recorded on three cine loop video clips of 4 seconds each at each ultrasound examination. 2.5. Metabolic and hormonal parameters The following blood metabolites were measured: IGF-I, GPX, P, Mg, and Ca, as well as BHB, NEFA, and urea. Plasma IGF-I concentrations were determined using a validated double-antibody RIA after ethanol–acetone–acetic acid extraction [23]. Recombinant IGF-I (Upstate, Millipore, Temecula, CA, USA) was used as tracer and standard, anti-human IGF-I (NHPP-NIDDK AFP 4892898; National Hormone and Peptide Program, Torrance, CA, USA; dilution 1:750,000) as the primary antibody and anti-rabbit IgG (Immunodiagnostic Systems, Boldon, UK) as the secondary antibody. The sensitivity of the IGF-I assay was 6 ng/mL. Intraassay CV for IGF-I were 16.1%, 11.1%, and 11.2% for control sera samples containing mean concentrations of 64.5, 106.9, and 491.5 ng IGF-I/mL, respectively. Interassay CV for the same control sera were 11.0%, 11.1%, and 11.2%, respectively. Urea, NEFA, BHB, GPX, Ca, Mg, and P were measured using a Randox RX Imola multichannel autoanalyzer (Randox Laboratories Ltd., Crumlin, Co. Antrim, Northern Ireland). Plasma BHB, NEFA, and urea concentrations were measured on an operationally enhanced random access (OPERA) analyzer (Bayer, Newbury, Berks, UK) using kinetic enzymatic kits (NEFA test kit, BHB RANBUT D-3-hydroxybutyrate test kit, urea test kit; Randox Laboratories Ltd., Co. Antrim, Northern Ireland). The minimum detectable concentration for urea was 0.51 mmol/L and the intraassay CV were 4.46%, 4.37%, and 1.58% for high-, medium-, and low-quality control sera, respectively. The minimum detectable concentration for NEFA was 0.04 mmol/L, and the intraassay CV was 4.85%, 0.99%, and 1.30% for high-, medium-, and low-quality control sera, respectively. The minimum detectable concentration for BHB was 0.07 mmol/L, and the intraassay CV was 3.32%, 0.99%, and 0.92% for high-, medium-, and low-quality control sera, respectively. GPX activity was determined using the Randox RX Imola Ransel kit. Samples were diluted 50 mL with 2 mL of diluting reagent before the start of the assays. Briefly, GPX catalyzed the oxidation of glutathione by cumene hydroperoxidase. In the presence of glutathione reductase and NADPH, the
oxidized glutathione (GSSG) was immediately converted to the reduced form with a concomitant oxidation of NADPH to NADPþ. This procedure gives a value in U/L of hemolysate. To get the value in U/mL packed cell volume, the value is multiplied by 61/1000 and subsequently divided by the value for packed cell volume. The minimum detectable concentration for GPX was 82.86 U/L, and the intraassay CV was 7.5%, 2.9%, and 4.2% for high-, medium-, and lowquality control sera, respectively. The concentrations of Ca and Mg were determined by a colorimetric method as per the manufacturer’s instructions. The minimum detectable concentration for Ca was 0.01 mmol/L, and the intraassay CV was 4.35%, 2.99%, and 2.16% for high-, medium-, and low-quality control sera, respectively. The minimum detectable concentration for Mg was 0.208 mmol/L, and the intra–assay CV was 3.89%, 1.97%, and 2.22% for high-, medium-, and low-quality control sera, respectively. Inorganic P was determined by UV method as per the manufacturer’s instructions. The minimum detectable concentration for P was 0.144 mmol/L, and the intraassay CV was 2.34%, 2.93%, and 2.82% for high-, medium-, and low-quality control sera, respectively. 2.6. Statistical analyses 2.6.1. Analysis of continuous data Metabolic parameters were classified as continuous variables. All variables were checked for normality and homogeneity of variance both visually and analytically using histograms, quantile–quantile (Q-Q) plots, and formal statistical tests in the UNIVARIATE procedures of SAS (version 9.3). For variables with a nonnormal distribution, the Box–Cox methodology was used to identify the most appropriate transformation. Continuous variables were analyzed using mixed models, with farm, cow identity, lactation, response, and calving status as fixed effects and time point as a repeated effect. Biologically plausible interactions were tested for significance in the model for each dependent variable. Fixed effects (P > 0.05) and interactions (P > 0.10) not associated with the dependent variables were removed by backward elimination with the exception of response and pregnancy status, which were forced into each model. All results are reported as least squares means and SE of the means for untransformed and 95% CI for log-transformed data. 2.6.2. Analysis of binary and ordinal data The occurrence of UI, clinically lame, NEB, AA, SCC 200,000, and healthy animals were classified as binary variables as well as the response to the synchrony protocol and the pregnancy status. The logit of the probability of a positive outcome for these variables was evaluated using logistic regression (PROC LOGISTIC), odds ratio (OR), and confidence intervals 95% (CI 95%) were calculated. Lactation, farm, response to synchronization, and pregnancy status were included as factors in the model, and calving day of the year and DIM were included as covariates to address possible confounding effects that these factors may have had on estrous response and pregnancy status. All biologically plausible interactions were also tested in the
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models. Main effects with P 0.05 for the likelihood ratio chi-square statistics were considered to contribute significantly to the model and were retained; estrus response rate and pregnancy status were forced into all models. 3. Results 3.1. Results summary The distributions of animals across the different disease categories are displayed in Table 2. A total of 159 animals were classified as having UI (39.6%), with 52 animals falling into the NEB category (12.9%), 56 in the clinically lame category (13.9%), 39 in the SCC category (9.7%), 21 in the AA category (5.2%), and the remainder of the animals were clarified as healthy (n ¼ 170, 42.3%). Overall, 83.6% (n ¼ 336) responded in estrus (were visually detected in estrus after the CIDR removal with the aid of tail paint) to the protocol; 42% (n ¼ 141) of the respondents were confirmed as pregnant 32 to 35 days after AI. The distribution of the cows that responded in estrus over the different disease categories is displayed in Table 2.
3.2. Uterine infection There were 748 uterine mucus samples collected with the aid of a metricheck device across two time points, 23 5 and 32 4 days postpartum. Determined by combined time points, 14.8% had clear or translucent vaginal mucus (score 0), 59.0% had clear mucus with flecks of pus (score 1), 19.0% had mucopurulent mucus (score 2), and 7.2% had purulent mucus (score 3). On the basis of the presence of mucopurulent or purulent mucus, the overall prevalence of UI was 26.2%, and there was no difference between sample days (27.7% vs 24.7%, P ¼ 0.59). The vaginal mucus had a fetid odor in 7.5% of cases, and there was no difference in the prevalence on Day 23 5 compared with Day 33 4 (7.9% vs 7.1%, P ¼ 0.54). Animals without a UI after calving were 1.9 times and two times more likely to respond to the CIDR protocol and be confirmed pregnant than those with a UI, respectively (1.5–2.4 CI, P 0.05; and 2.1–1.7 CI, P 0.05). For the cows with UIs, 77.4% responded in estrus to the protocol and 32.5% of the animals that
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responded were confirmed pregnant by ultrasonography on Day 32 to 35 after AI (P 0.001). Ultrasonography scans (n ¼ 697) were also recorded at the same time as the two mucus scores. The animals were sorted into four groups (0 to 3) according to their ultrasonography score. There was no detectable fluid in the uterine horns in 39.6% of animals and included both cyclic and noncyclic cows (score ¼ 0), 36.2% of animals had a mild inflammation of the uterine horns (score ¼ 1), 19% had an infection of the uterine tract (endometritis) (score ¼ 2), and 5.3% of the animals had abnormal corpus luteum development and ovarian cysts (score ¼ 3). The animals with a uterine scan score 2 were 1.7 times and 1.5 times more likely not to respond or be nonpregnant than those animals with a uterine score of 0.05 0.01 >0.05 0.01 >0.05 0.01 >0.05 >0.05 0.01 >0.05 >0.05 0.01 0.01 >0.05 0.01 >0.05 >0.05 >0.05 0.01 >0.05
0.47–1.28 1.36–2.19 1.05–2.08 1.46–2.78 1.00–2.09 1.39–2.24 0.75–1.29 0.86–1.42 2.17–5.30 0.84–1.4 0.52–1.17 1.35–2.59 2.85–6.94 0.93–2.01 1.75–4.74 0.56–1.53 0.62–1.38 0.76–1.31 1.298–3.37 0.96–1.92
Abbreviations: AA, anovulatory anestrus; ClinLame, clinically lame; NEB, negative energy balance; SCC, somatic cell count 200,000; UI, uterine infection.
J.C. McNally et al. / Theriogenology xxx (2014) 1–10
well as the probability of them subsequently becoming pregnant was associated with the animal falling into one or more categories (Fig. 3). 3.9. Metabolic profiles The response to the estrous synchronization protocol was significant across the relative time points for the IGF-I and urea parameters (P < 0.0001) but not for GPX, BHB, and NEFA concentrations (Tables 4 and 5). In Table 5, Ca was the only parameter that had a significant effect between those that responded in estrus and those animals that did not respond to the protocol (1.05 and 0.96, P ¼ 0.0001). Cows that suffered low Ca immediately after calving had no difference in their conception rate than those with a healthy Ca level (P 0.05). Animals outside the normal blood magnesium (Mg) concentration range of 0.8 and 1.3 mmol/L in the first 48 hours had a lower estrous response rate to the protocol than the healthy animals (100% vs 82%, P 0.05), although the number of animals confirmed pregnant was not significant. Phosphorus levels on the day of calving and Day 1 had no effect on the response or pregnancy rate (P 0.05), and although GPX had no effect on the response to the protocol, it had an effect on the pregnancy rate (P 0.05). 4. Discussion In the present study, 26.2% of animals had mucopurulent or purulent mucus in the vagina. Animals without a UI by metricheck examination were twice as likely to be pregnant after the synchronization protocol as those animals with an infection. In the study by LeBlanc et al. [24], the reproductive performance of cows with purulent discharge on examination was significantly lower than that of cows with no abnormal discharge. Uterine infection suppresses pituitary LH secretion, perturbs postpartum ovarian follicle growth, and disrupts ovulation in cattle [25–27]. Thus, UI is associated with lower conception rates, increased intervals from calving to first service or conception, and more culls for failure to conceive [24,28,29].
Fig. 3. The probability of cows responding in estrus (P < 0.0001) to the CIDR synchronization protocol and subsequently confirmed pregnant (P < 0.0001) depending the number of physiological or diseases classifications they were in. Only cows that responded in estrus are included in the conception figures. The number of animals within each group is also presented.
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Animals that had an ultrasonography score of 2 also had a significantly reduced estrous response and conception rates after progesterone synchronization. A metricheck examination to detect abnormal uterine discharge is a more sensitive and specific method for detection of endometritis than transrectal palpation [24,30]. However, metricheck examinations often fail to identify all cows that are truly at risk of poor reproductive performance [31]. Clinically, the challenge is to identify all cows that are truly at risk of impaired fertility. Postpartum endometritis (UI) was characterized clinically by the presence of pus in the vagina [32]. Animals with NEB had a longer interval to first ovulation [33]; therefore, the CIDR protocol had a positive response on those animals within the NEB classification. Cows in early lactation are prone to NEB, which arises from high milk energy output and relatively low feed intake. In this study, those animals classified as having NEB had a positive response rate to the synchronization but a negative outcome regarding the conception rate. The association between BCS loss in early lactation and subsequent reproductive performance is now well established [34–36]. During the period of declining NEB, LH pulses are suppressed and dominant follicles (DFs) that develop have a decreased chance of producing sufficient estradiol to induce a preovulatory gonadotropin surge [37,38]. Beta-hydroxybutyrate and NEFA blood levels are also used to calculate if an animal is experiencing NEB. This strategy is based on the use of BHB for the monitoring of subclinical ketosis in lactating cows with suggested optimal sampling times between 5 and 50 days in milk and the use of NEFA testing for the detection of prepartum NEB and fatty liver in cows that are from 2 to 14 days pre-calving. In this study, the cows with BCS loss of 0.5 did not correlate with those in NEB according to the blood analysis (33%), the reason for this may be from the time of sampling within a day as it has a critical influence on the outcome of blood metabolite analysis [39]. For BHB, the ideal time for sampling is suggested as 4 to 5 hours after feeding, whereas for NEFA just before feeding was recommended; in this study all animals were sampled after morning milking regardless of feeding time. In this study, 14.3% of animals were regarded as clinically lame within 37 days postpartum; this had an effect on the response to the synchronization protocol and the conception rate. More than 65% of the annual incidence of lameness occurs within the first month of lactation [40]. One explanation for this might be the changes occurring during the transition period (21 days before and after calving), such as new environment (type of floor surface, bedding), feeding management, and nutrition [41]. The failure to ovulate is associated with a reduced LH pulse frequency, lower estradiol concentrations or responsiveness to estradiol, and the absence of an LH surge [42], hence explaining the low conception rate (22.7%) within this group. In this study, we had a low number of animals that could be classified as having only AA or only a raised SCC. However, we still think that it is important to report on the impact of these conditions on response to synchronization. The animals with an SCC 200,000 cells/mL were grouped
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Table 4 The effect of metabolic parameters at sample points relative to calving on the response status after synchronization on 402 cows (data are means with the pooled SEM). Blood
Pre-calvinga No response
GPX IGF-Ib BHBb NEFAb UREAb
151.3 171.48c 0.56 0.29 3.45c
First post-calvinga
Second post-calvinga
Responded
No response
Responded
No response
Responded
156.37 172.46c 0.54 0.26 3.09d
155.88 67.85d 1.16 0.57 4.08e
160.26 78.89e 0.98 0.48 4.14e
159.06 77.14e,f 1.09 0.47 4.16e,f
162.29 85.86f 0.93 0.46 4.41e
SEM
2.18 3.63 0.03 0.02 0.08
P values Response
Time point
Response time
0.22 0.09 0.29 0.68 0.54
0.0002
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Theriogenology journal homepage: www.theriojournal.com
Effects of physiological and/or disease status on the response of postpartum dairy cows to synchronization of estrus using an intravaginal progesterone device Julie C. McNally a, Mark A. Crowe a, b, *, James F. Roche a, Marijke E. Beltman a a b
School of Veterinary Medicine, University College Dublin, Belfield, Dublin, Ireland Conway Institute for Biomolecular and Biomedical Research, University College Dublin, Belfield, Dublin, Ireland
a r t i c l e i n f o
a b s t r a c t
Article history: Received 26 May 2014 Received in revised form 13 August 2014 Accepted 13 August 2014
Progesterone treatments are used to increase submission rates in postpartum dairy cows; however, in many cases the protocol is used as a blanket therapy for all cows without regard for physiological or disease state. The objective of this study was to identify the physiological or disease classes of cows that respond well (or not) to synchronization of estrus via progesterone. Dairy cows (n ¼ 402) were monitored peri and postpartum to establish their physiological or disease status. Animals were classified as having negative energy balance, clinical lameness, uterine infection (UI), anovulatory anestrus, high somatic cell counts, and healthy (H). Blood samples were collected at five different time points and analyzed for metabolites. All animals received an 8-day controlled internal drug release protocol, which included GnRH at insertion and PGF2a the day before removal. Response to the protocol was determined by visual observation of estrus synchronization. Conception rate was determined by ultrasonography between Days 32 and 35 after artificial insemination. Animals without UI were 1.9 times more likely to respond and two times more likely to be confirmed pregnant than those with UI. There was no relationship between negative energy balance and clinical lameness in the visual estrous response, but both conditions were associated with reduced conception rates. Dairy cows in anovulatory anestrus responded successfully to the protocol in both estrous response and conception rates. High glutathione peroxidase concentrations had a positive effect on conception rates, whereas high non-esterified fatty acids and beta-hydroxybutyrate had a negative effect on the estrous response. In conclusion, disease and physiological states of dairy cows determined the response to progesterone-based synchronization. The more disease or physiological problems the cows had, the lower the estrous response and conception rates; cows with these problems were not ideal candidates for synchronization. Both anestrus and healthy dairy cows were good responders to progesterone-based synchronization. Ó 2014 Elsevier Inc. All rights reserved.
Keywords: Dairy cattle Estrous synchronization Physiological state Disease
1. Introduction Modern dairy production is based on high-yielding dairy cows producing over 9000 kg per lactation. One of
* Corresponding author. Tel.: þ353 1 716 6255; fax: þ353 (0)1 716 6255. E-mail address: [email protected] (M.A. Crowe). 0093-691X/$ – see front matter Ó 2014 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.theriogenology.2014.08.006
the key problem areas decreasing the efficiency and sustainability of milk production is infertility. In Irish dairy herds, cow fertility measured as calving rate to a single insemination declined at a rate of almost 1% per annum (55% to 44%) in the period from 1990 to 2001 [1]. Infertility has large economic impacts, and the importance of infertility for dairy farmers was emphasized by the results of a recent Delphi study [2] where infertility was one of the main concerns of farmers. Cows selected for high milk
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production may be more prone to an increased incidence of diseases such as endometritis, mastitis, milk fever, and lameness, as well as a higher risk of going into negative energy balance (NEB) postpartum, all of which have a negative effect on their reproductive status [3,4]. Apart from the reduced conception rates, some of the key causes of low reproductive efficiency in postpartum high-yielding dairy cows are as follows: (1) an increased incidence of clinical or subclinical endometritis, (2) a larger postpartum interval to first ovulation, (3) increased incidence of irregular cycles, (4) shorter duration and less intense expression of estrus synchronization, and (5) reduced conception rate per single insemination often followed by an increase in fetal loss [5]. The factors contributing to infertility problems in postpartum dairy cows are multifactorial and include genetics, nutrition, health, welfare, and poor reproductive management. Reproductive performance influences profitability of a dairy cow–calf operation. A high reproductive performance is especially important for seasonal dairy herds based on pasture where breeding and calving are restricted to a limited period of the year in order to match the feed requirements to the seasonal pattern of pasture growth [6]. The ability to synchronize onset of estrus, and hence the time of breeding and calving, offers potential economic and management benefits to dairy farmers. It increases the rate of genetic gain through more usage of artificial insemination (AI) with genetically superior proven sires with high-accuracy expected progeny differences, increases milk production through advancement of the mean calving date, and reduces the percentage of nonpregnant cows at the end of the breeding season [6]. The transition period of 3 weeks pre-calving until 3 weeks post-calving is associated with a peak incidence of production diseases that may disrupt animal fertility. Although traditionally regarded as the significant metabolic disorders of dairy cows (hypocalcemia, hypomagnesemia, and ketosis), the term production disease, which is influenced by high production and management of the transition cow, has been broadened to include conditions such as retained placenta, uterine discharge, milk fever, mastitis, and lameness [7,8]. The precise cause of impaired immune function in transition cows is unclear, although the peripartum decrease in energy, vitamin, and mineral intake, which can result in NEB and mobilization of body fat are contributing factors. The dramatic changes in progesterone and estrogen levels in late gestation also seem to play a role [9–11]. Lame cows have a lower overall pregnancy rate and 65% of the annual incidence of lameness occurs within the first month of lactation; therefore, lameness is one of the major reasons for culling in dairy herds [12]. Postpartum uterine disease is also a component of reproductive performance [13]. Butler and Smith [14] found that cows that lost less than 0.5 units of body condition score (BCS) during the first 5 weeks postpartum had higher conception rates at the first service than cows that lost more than 0.5 BCS. Clinical mastitis or high somatic cell count (SCC) in the postpartum period delays the resumption of ovarian activity considerably [15,16]. Our hypothesis was that the response to progesterone-based synchronization in dairy cows is
dependent on the physiological and disease status of the animal in the postpartum period. Both progestagens and prostaglandins are used routinely for estrous synchronization in cattle. Progesterone-based methods to synchronize estrus result in less variation in the timing of onset of estrus compared with prostaglandinbased methods [17], and they may be used to induce estrus in anestrous cows in the postpartum period. In the current study, an 8-day progesterone-based treatment, which included GnRH at the insertion and PGF2a the day before removal, was used. The objective of this experiment was to determine the response to progesterone-based synchronization in dairy cows in relation to their physiological and disease status with the aim to identify the physiological classes of cows that respond well (or do not respond well) to a synchronization protocol including progesterone. 2. Materials and methods 2.1. Study animals All experimental procedures involving animals were licensed in accordance with the Cruelty to Animals Act (Ireland 1876) and the European Community Directive 86/ 609/EC and were sanctioned by the University College Dublin Animal Research Ethics Committee. This study was conducted using multiparous (N ¼ 250) and primiparous cows (N ¼ 152) from five commercial spring-calving dairy herds in the Leinster region (Ireland) between November 2011 and July 2012. Cows of all parities (1 to 11) were represented; median lactation number was 2 and maximum was 11, with normal (no intervention, calving on own) and low degrees (minimal intervention by the farmer) of calving difficulty that calved between January and April 2012 were enrolled in the trial. All animals were housed indoors within a free-stall barn during their 60 days dry period before calving. After parturition, they remained indoors within a free-stall barn until turn out to pasture in spring (varied from early February to April across the farms) with the exception of one herd that remained indoors throughout the study. Breeds represented were Holstein (n ¼ 100), Holstein-Friesian (n ¼ 203), and Crossbreds (n ¼ 99). The distribution of breeds on individual farms ranged from primarily HolsteinFriesian to primarily crossbreds. 2.2. Experimental procedures All cows enrolled on the trial were evaluated at seven time points around calving. At each sample/evaluation time, locomotion score (LM), BCS, rectal temperature as a measure of overall health, and vaginal mucus score (MS) as a measurement of uterine health were recorded. In addition, blood samples for metabolic parameters were collected. Insulin-like growth factor-I (IGF-I), glutathione peroxidase (GPX; as an estimate of the selenium status) were analyzed at five time points, beta-hydroxybutyrate (BHB), nonesterified fatty acids (NEFAs), and urea were analyzed at three time points, calcium (Ca), magnesium (Mg), and phosphorus (P) were analyzed at one time point. A timeline of these seven time points is represented in Table 1.
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postpartum. Animals classified as NEB had a loss of 0.5 of a BCS from calving to Day 32 4 postpartum. Animals that had a locomotion score of 3 post-calving from Days 0 to 32 4 were classified as clinically lame [18]. The animals classified in the SSC 200,000 cells/mL were defined as those having an SCC 200,000 cells/mL 30 days before breeding. Animals classified as anovulatory anestrus (AA) had clean uterine horns with no evidence of inflammation or infection but had delayed ovulation (absence of detectable CL when ovaries were scanned up to Day 37 9 and at controlled internal drug release (CIDR) insertion). The animals that were in none of the above classifications were then categorized as healthy (H). All herds were tested for Johnes, bovine viral diarrhea, infectious bovine rhinotracheitis, leptospirosis, liver, and rumen flukes through a bulk milk sample. This sample was taken when all the cows on the trial were postpartum and tested using an ELISA bulk milk sampling kit. Individual milk recording for SCC took place by a commercial company four times during the trial period. Body temperature was recorded on Day 10 4 postpartum using a digital rectal thermometer.
Locomotion was scored as described by Spreacher et al. [16], where a score of 1 represented normal locomotion and 2 represented mild lameness; these animals showed an arched back while walking, but their gait remained normal. An animal with a locomotion score of 3 displayed an arched back when both standing and walking and an animal with a score of 4 showed an arched back while standing and walking and a gait that is one deliberate step at a time. Body condition was scored on a 5-point scale with increments of 0.25 (as described by Edmonson et al. [19]). Blood samples were taken at five time points (Days 7 7 pre-calving, 4 4, 18 4, 25 3, and 32 4 postpartum) by coccygeal venipunture. Blood samples were collected into three Vacutainer tubes (Becton, Dickinson and Company, Plymouth, UK), two containing lithium heparin for plasma collection and one containing a clot activator for serum collection. At each blood time point, a plasma and serum sample was stored at ambient temperature for a maximum of 6 hours, and then at 4 C for a further 18 hours; each sample was decanted after centrifugation for 20 minutes at 1600 g and stored at 20 C until subsequent analysis. A plasma sample was assayed for GPX within 24 hours of collection. Vaginal mucus was collected by means of a metricheck (Simcro Tech, Hamilton, New Zealand) device that scoops discharge from the anterior vagina. The mucus was assessed by one person for color, proportion, and volume of discharge, and a character score assigned as follows: (0) clear or translucent mucus; (1) mucus containing flecks of white or off-white pus; (2) less than 50 mL exudates containing less than 50% white or off-white mucopurulent material; and (3) more than 50 mL exudates containing purulent material, usually white or yellow, but occasionally sanguineous [20]. The vaginal mucus was also assessed by odor, and given a score 0 for normal odor or a score of 1 if a fetid odor was detected [21]. The metricheck device was cleaned and placed in suitable disinfectant between each cow sampled. The mucus scoring was carried out at three time points postpartum (Days 18 4, 23 5, and 32 4). The SCC of each animal was recorded monthly, and animals were assigned to a grade according to their SCC score. Grade 1 was animals with a score less than200,000; grade 2 animals had an SCC 200,000. Animals were assigned to a classification according to their disease or physiological state. Animals were assigned to the uterine infection (UI) classification when the metricheck score was 2 [21] on Days 23 5 and 32 4
2.3. Synchronization treatments and AI Estrous cycles of all animals were synchronized using a CIDR (containing 1.38 g progesterone, Pfizer Animal Health, Dublin, Ireland; now Zoetis) intravaginally for 8 days, with a 2-mL im injection of a GnRH (Receptal, MSD Animal Health, Ireland) at the time of CIDR insertion and a 2.5-mL im injection of PGF2a analogue (Estrumate, MSD Animal Health) 1 day before CIDR withdrawal. All cows were 37 days in milk (DIM) (mean ¼ 59; range 37–93 DIM) at the initiation of synchrony treatment, resulting in synchronized estrus or ovulation at 47 DIM (mean ¼ 69; range ¼ 47–103 DIM). Cows were inseminated by the AM/ PM rule (a cow observed in standing estrus in the morning was inseminated in the afternoon of the same day and a cow observed in standing estrus in the afternoon or evening was bred the following morning), following detection of estrus with the aid of tail paint. Detection of estrus was performed four times per 24-hour period for 20 minutes (i.e., 6-hour intervals). All inseminations were performed by experienced technicians from commercial AI companies or by the herd owners and farm staff licensed by the Department of Agriculture, Fisheries and Food (Ireland), to carry out AI.
Table 1 Timeline of key sample and data collection points relative to day of calving for dairy cows enrolled in the study. Time point
Days from calving
BCS
LM
1 2 3 4 5 6 7
60 to 40 21 to 0 0–7 7–14 14–21 21–28 28–35 w50
U U U
U
Mucus
Temperature
U
Serum
Plasma
U U
U U
U U U
U U U
Scan
U
U CIDR insertion
U U U
Abbreviations: BCS, body condition score; CIDR, controlled internal drug release; LM, locomotion score.
U U U
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2.4. Transrectal ultrasonography The reproductive tracts of all cows were examined by transrectal ultrasound on Days 25 3 and 32 4 postcalving and immediately before initiation of synchronization. One experienced operator using an Ibex Lite ultrasound scanner (Ibex Lite; E.I Medical Imaging, CO, USA) equipped with a 5-MHz transrectal transducer performed the examinations. Cows were assigned an ultrasound reproductive tract score describing the volume and echogenicity of fluid contained within the uterus [22]. All cows were scanned at 32 to 35 days after AI to confirm pregnancy; this was confirmed by palpation per rectum, visualization of a fluid-filled uterine horn, and the presence of a conceptus as positive indicators of pregnancy. The uterine, ovarian, and pregnancy findings were recorded on three cine loop video clips of 4 seconds each at each ultrasound examination. 2.5. Metabolic and hormonal parameters The following blood metabolites were measured: IGF-I, GPX, P, Mg, and Ca, as well as BHB, NEFA, and urea. Plasma IGF-I concentrations were determined using a validated double-antibody RIA after ethanol–acetone–acetic acid extraction [23]. Recombinant IGF-I (Upstate, Millipore, Temecula, CA, USA) was used as tracer and standard, anti-human IGF-I (NHPP-NIDDK AFP 4892898; National Hormone and Peptide Program, Torrance, CA, USA; dilution 1:750,000) as the primary antibody and anti-rabbit IgG (Immunodiagnostic Systems, Boldon, UK) as the secondary antibody. The sensitivity of the IGF-I assay was 6 ng/mL. Intraassay CV for IGF-I were 16.1%, 11.1%, and 11.2% for control sera samples containing mean concentrations of 64.5, 106.9, and 491.5 ng IGF-I/mL, respectively. Interassay CV for the same control sera were 11.0%, 11.1%, and 11.2%, respectively. Urea, NEFA, BHB, GPX, Ca, Mg, and P were measured using a Randox RX Imola multichannel autoanalyzer (Randox Laboratories Ltd., Crumlin, Co. Antrim, Northern Ireland). Plasma BHB, NEFA, and urea concentrations were measured on an operationally enhanced random access (OPERA) analyzer (Bayer, Newbury, Berks, UK) using kinetic enzymatic kits (NEFA test kit, BHB RANBUT D-3-hydroxybutyrate test kit, urea test kit; Randox Laboratories Ltd., Co. Antrim, Northern Ireland). The minimum detectable concentration for urea was 0.51 mmol/L and the intraassay CV were 4.46%, 4.37%, and 1.58% for high-, medium-, and low-quality control sera, respectively. The minimum detectable concentration for NEFA was 0.04 mmol/L, and the intraassay CV was 4.85%, 0.99%, and 1.30% for high-, medium-, and low-quality control sera, respectively. The minimum detectable concentration for BHB was 0.07 mmol/L, and the intraassay CV was 3.32%, 0.99%, and 0.92% for high-, medium-, and low-quality control sera, respectively. GPX activity was determined using the Randox RX Imola Ransel kit. Samples were diluted 50 mL with 2 mL of diluting reagent before the start of the assays. Briefly, GPX catalyzed the oxidation of glutathione by cumene hydroperoxidase. In the presence of glutathione reductase and NADPH, the
oxidized glutathione (GSSG) was immediately converted to the reduced form with a concomitant oxidation of NADPH to NADPþ. This procedure gives a value in U/L of hemolysate. To get the value in U/mL packed cell volume, the value is multiplied by 61/1000 and subsequently divided by the value for packed cell volume. The minimum detectable concentration for GPX was 82.86 U/L, and the intraassay CV was 7.5%, 2.9%, and 4.2% for high-, medium-, and lowquality control sera, respectively. The concentrations of Ca and Mg were determined by a colorimetric method as per the manufacturer’s instructions. The minimum detectable concentration for Ca was 0.01 mmol/L, and the intraassay CV was 4.35%, 2.99%, and 2.16% for high-, medium-, and low-quality control sera, respectively. The minimum detectable concentration for Mg was 0.208 mmol/L, and the intra–assay CV was 3.89%, 1.97%, and 2.22% for high-, medium-, and low-quality control sera, respectively. Inorganic P was determined by UV method as per the manufacturer’s instructions. The minimum detectable concentration for P was 0.144 mmol/L, and the intraassay CV was 2.34%, 2.93%, and 2.82% for high-, medium-, and low-quality control sera, respectively. 2.6. Statistical analyses 2.6.1. Analysis of continuous data Metabolic parameters were classified as continuous variables. All variables were checked for normality and homogeneity of variance both visually and analytically using histograms, quantile–quantile (Q-Q) plots, and formal statistical tests in the UNIVARIATE procedures of SAS (version 9.3). For variables with a nonnormal distribution, the Box–Cox methodology was used to identify the most appropriate transformation. Continuous variables were analyzed using mixed models, with farm, cow identity, lactation, response, and calving status as fixed effects and time point as a repeated effect. Biologically plausible interactions were tested for significance in the model for each dependent variable. Fixed effects (P > 0.05) and interactions (P > 0.10) not associated with the dependent variables were removed by backward elimination with the exception of response and pregnancy status, which were forced into each model. All results are reported as least squares means and SE of the means for untransformed and 95% CI for log-transformed data. 2.6.2. Analysis of binary and ordinal data The occurrence of UI, clinically lame, NEB, AA, SCC 200,000, and healthy animals were classified as binary variables as well as the response to the synchrony protocol and the pregnancy status. The logit of the probability of a positive outcome for these variables was evaluated using logistic regression (PROC LOGISTIC), odds ratio (OR), and confidence intervals 95% (CI 95%) were calculated. Lactation, farm, response to synchronization, and pregnancy status were included as factors in the model, and calving day of the year and DIM were included as covariates to address possible confounding effects that these factors may have had on estrous response and pregnancy status. All biologically plausible interactions were also tested in the
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models. Main effects with P 0.05 for the likelihood ratio chi-square statistics were considered to contribute significantly to the model and were retained; estrus response rate and pregnancy status were forced into all models. 3. Results 3.1. Results summary The distributions of animals across the different disease categories are displayed in Table 2. A total of 159 animals were classified as having UI (39.6%), with 52 animals falling into the NEB category (12.9%), 56 in the clinically lame category (13.9%), 39 in the SCC category (9.7%), 21 in the AA category (5.2%), and the remainder of the animals were clarified as healthy (n ¼ 170, 42.3%). Overall, 83.6% (n ¼ 336) responded in estrus (were visually detected in estrus after the CIDR removal with the aid of tail paint) to the protocol; 42% (n ¼ 141) of the respondents were confirmed as pregnant 32 to 35 days after AI. The distribution of the cows that responded in estrus over the different disease categories is displayed in Table 2.
3.2. Uterine infection There were 748 uterine mucus samples collected with the aid of a metricheck device across two time points, 23 5 and 32 4 days postpartum. Determined by combined time points, 14.8% had clear or translucent vaginal mucus (score 0), 59.0% had clear mucus with flecks of pus (score 1), 19.0% had mucopurulent mucus (score 2), and 7.2% had purulent mucus (score 3). On the basis of the presence of mucopurulent or purulent mucus, the overall prevalence of UI was 26.2%, and there was no difference between sample days (27.7% vs 24.7%, P ¼ 0.59). The vaginal mucus had a fetid odor in 7.5% of cases, and there was no difference in the prevalence on Day 23 5 compared with Day 33 4 (7.9% vs 7.1%, P ¼ 0.54). Animals without a UI after calving were 1.9 times and two times more likely to respond to the CIDR protocol and be confirmed pregnant than those with a UI, respectively (1.5–2.4 CI, P 0.05; and 2.1–1.7 CI, P 0.05). For the cows with UIs, 77.4% responded in estrus to the protocol and 32.5% of the animals that
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responded were confirmed pregnant by ultrasonography on Day 32 to 35 after AI (P 0.001). Ultrasonography scans (n ¼ 697) were also recorded at the same time as the two mucus scores. The animals were sorted into four groups (0 to 3) according to their ultrasonography score. There was no detectable fluid in the uterine horns in 39.6% of animals and included both cyclic and noncyclic cows (score ¼ 0), 36.2% of animals had a mild inflammation of the uterine horns (score ¼ 1), 19% had an infection of the uterine tract (endometritis) (score ¼ 2), and 5.3% of the animals had abnormal corpus luteum development and ovarian cysts (score ¼ 3). The animals with a uterine scan score 2 were 1.7 times and 1.5 times more likely not to respond or be nonpregnant than those animals with a uterine score of 0.05 0.01 >0.05 0.01 >0.05 0.01 >0.05 >0.05 0.01 >0.05 >0.05 0.01 0.01 >0.05 0.01 >0.05 >0.05 >0.05 0.01 >0.05
0.47–1.28 1.36–2.19 1.05–2.08 1.46–2.78 1.00–2.09 1.39–2.24 0.75–1.29 0.86–1.42 2.17–5.30 0.84–1.4 0.52–1.17 1.35–2.59 2.85–6.94 0.93–2.01 1.75–4.74 0.56–1.53 0.62–1.38 0.76–1.31 1.298–3.37 0.96–1.92
Abbreviations: AA, anovulatory anestrus; ClinLame, clinically lame; NEB, negative energy balance; SCC, somatic cell count 200,000; UI, uterine infection.
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well as the probability of them subsequently becoming pregnant was associated with the animal falling into one or more categories (Fig. 3). 3.9. Metabolic profiles The response to the estrous synchronization protocol was significant across the relative time points for the IGF-I and urea parameters (P < 0.0001) but not for GPX, BHB, and NEFA concentrations (Tables 4 and 5). In Table 5, Ca was the only parameter that had a significant effect between those that responded in estrus and those animals that did not respond to the protocol (1.05 and 0.96, P ¼ 0.0001). Cows that suffered low Ca immediately after calving had no difference in their conception rate than those with a healthy Ca level (P 0.05). Animals outside the normal blood magnesium (Mg) concentration range of 0.8 and 1.3 mmol/L in the first 48 hours had a lower estrous response rate to the protocol than the healthy animals (100% vs 82%, P 0.05), although the number of animals confirmed pregnant was not significant. Phosphorus levels on the day of calving and Day 1 had no effect on the response or pregnancy rate (P 0.05), and although GPX had no effect on the response to the protocol, it had an effect on the pregnancy rate (P 0.05). 4. Discussion In the present study, 26.2% of animals had mucopurulent or purulent mucus in the vagina. Animals without a UI by metricheck examination were twice as likely to be pregnant after the synchronization protocol as those animals with an infection. In the study by LeBlanc et al. [24], the reproductive performance of cows with purulent discharge on examination was significantly lower than that of cows with no abnormal discharge. Uterine infection suppresses pituitary LH secretion, perturbs postpartum ovarian follicle growth, and disrupts ovulation in cattle [25–27]. Thus, UI is associated with lower conception rates, increased intervals from calving to first service or conception, and more culls for failure to conceive [24,28,29].
Fig. 3. The probability of cows responding in estrus (P < 0.0001) to the CIDR synchronization protocol and subsequently confirmed pregnant (P < 0.0001) depending the number of physiological or diseases classifications they were in. Only cows that responded in estrus are included in the conception figures. The number of animals within each group is also presented.
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Animals that had an ultrasonography score of 2 also had a significantly reduced estrous response and conception rates after progesterone synchronization. A metricheck examination to detect abnormal uterine discharge is a more sensitive and specific method for detection of endometritis than transrectal palpation [24,30]. However, metricheck examinations often fail to identify all cows that are truly at risk of poor reproductive performance [31]. Clinically, the challenge is to identify all cows that are truly at risk of impaired fertility. Postpartum endometritis (UI) was characterized clinically by the presence of pus in the vagina [32]. Animals with NEB had a longer interval to first ovulation [33]; therefore, the CIDR protocol had a positive response on those animals within the NEB classification. Cows in early lactation are prone to NEB, which arises from high milk energy output and relatively low feed intake. In this study, those animals classified as having NEB had a positive response rate to the synchronization but a negative outcome regarding the conception rate. The association between BCS loss in early lactation and subsequent reproductive performance is now well established [34–36]. During the period of declining NEB, LH pulses are suppressed and dominant follicles (DFs) that develop have a decreased chance of producing sufficient estradiol to induce a preovulatory gonadotropin surge [37,38]. Beta-hydroxybutyrate and NEFA blood levels are also used to calculate if an animal is experiencing NEB. This strategy is based on the use of BHB for the monitoring of subclinical ketosis in lactating cows with suggested optimal sampling times between 5 and 50 days in milk and the use of NEFA testing for the detection of prepartum NEB and fatty liver in cows that are from 2 to 14 days pre-calving. In this study, the cows with BCS loss of 0.5 did not correlate with those in NEB according to the blood analysis (33%), the reason for this may be from the time of sampling within a day as it has a critical influence on the outcome of blood metabolite analysis [39]. For BHB, the ideal time for sampling is suggested as 4 to 5 hours after feeding, whereas for NEFA just before feeding was recommended; in this study all animals were sampled after morning milking regardless of feeding time. In this study, 14.3% of animals were regarded as clinically lame within 37 days postpartum; this had an effect on the response to the synchronization protocol and the conception rate. More than 65% of the annual incidence of lameness occurs within the first month of lactation [40]. One explanation for this might be the changes occurring during the transition period (21 days before and after calving), such as new environment (type of floor surface, bedding), feeding management, and nutrition [41]. The failure to ovulate is associated with a reduced LH pulse frequency, lower estradiol concentrations or responsiveness to estradiol, and the absence of an LH surge [42], hence explaining the low conception rate (22.7%) within this group. In this study, we had a low number of animals that could be classified as having only AA or only a raised SCC. However, we still think that it is important to report on the impact of these conditions on response to synchronization. The animals with an SCC 200,000 cells/mL were grouped
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Table 4 The effect of metabolic parameters at sample points relative to calving on the response status after synchronization on 402 cows (data are means with the pooled SEM). Blood
Pre-calvinga No response
GPX IGF-Ib BHBb NEFAb UREAb
151.3 171.48c 0.56 0.29 3.45c
First post-calvinga
Second post-calvinga
Responded
No response
Responded
No response
Responded
156.37 172.46c 0.54 0.26 3.09d
155.88 67.85d 1.16 0.57 4.08e
160.26 78.89e 0.98 0.48 4.14e
159.06 77.14e,f 1.09 0.47 4.16e,f
162.29 85.86f 0.93 0.46 4.41e
SEM
2.18 3.63 0.03 0.02 0.08
P values Response
Time point
Response time
0.22 0.09 0.29 0.68 0.54
0.0002
