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Early pregnancy loss (EPL) between days 15-65 after breeding has been shown to occur in 7.9% of equine pregnancies with substantial economical, welfare and safety implications. Whilst maternal age has been recognised as an important risk factor in relation to the incidence of EPL, few other risk factors have been conclusively identified. Further, multivariable data analysis of risk factors for EPL is sparse. A prospective cohort investigation of thoroughbred broodmares in the United Kingdom was conducted over the 2013 and 2014 breeding seasons. Information relating to 28 factors including mare, stallion, pregnancy and therapeutic interventions was collected using questionnaires and entered into a custom-designed Microsoft Access database. Mixed effects logistic regression was used to determine risk factors for EPL, including 'mare' as a random effect to account for repeat pregnancies in the same mare. Stallion, stud and veterinarian were also evaluated as random effects. Variables with a p-value of0.25 in univariable analysis were taken forward for consideration in the multivariable model which was built using a forward stepwise approach. Data were collected on 2245 pregnancies in 1753 mares. Increasing mare age (OR = 1.11, 95% confidence interval (CI) = 1.04, 1.18, p = 0.001), having had one previous foal (OR = 3.52, 95% CI = 1.56, 7.95, p = 0.002) and presence of uterine cysts (OR = 1.76, 95% CI = 1.07, 2.91, p = 0.03) were all associated with increased odds of EPL following multivariable analysis. Increasing day 15/16 scan vesicle size (OR = 0.24, 95% CI = 0.16, 0.38, p 0.001) and the use of ovulatory induction agents (OR = 0.31, 95% CI = 0.17, 0.55, p 0.001) were negatively associated with EPL. Stallion, stud and veterinarian were not significantly associated with EPL. Analysis of a subpopulation of 344 multiple (twin and triplet) pregnancies found that the use of flunixin meglumine at the time of manual reduction of a multiple pregnancy resulted in reduced odds of EPL (OR = 0.34, 95% CI = 0.14, 0.84, p = 0.02). Results from this study can be used by stud farm personnel when assessing their broodmare population and by clinicians when deciding upon therapeutic strategies. Additional work can be focused around these risk factors to further our understanding of the pathophysiology of EPL.

The natural cervical relaxation which occurs at estrus in the ewe may be initiated by binding of hyaluronan (HA) to its receptor CD44. Indeed, we have previously shown that HA content and fragment size in the ovine cervix varies with the stage of the estrous cycle. Despite the importance of cervical relaxation in promoting sperm transport and facilitating the possible development of transcervical artificial insemination (AI), the mechanisms coordinating these changes in HA content remain to be defined. Hyaluronan synthases (HAS) 1, 2, and 3 regulate HA biosynthesis and herein, we describe the changing pattern of HAS isoform expression during the estrous cycle to determine whether this may underpin HA-mediated changes in relaxation of the ovine cervix. Accordingly, cervices were collected from 24 cyclic sheep (n = 8 / group) at the luteal, pre-luteinizing hormone (LH) and post-LH surge stages. Protein and mRNA expression for HAS 1, 2 and 3 was determined in five different tissue layers (epithelium, subepithelial stroma, and longitudinal, circular and transverse muscle) of the vaginal, mid and uterine regions of each cervix by immunohistochemistry and in situ hybridization, respectively. HA synthases were expressed in all the tissue layers and regions of the cervix, and the pattern of expression was similar for mRNA and protein. HAS1 protein and mRNA expression was significantly (P ≤ 0.05) higher at the pre-LH surge stage, while HAS 2 and 3 protein and mRNA expression was significantly (P ≤ 0.001) higher at the luteal stage. Overall, both HAS protein and mRNA expression was significantly (P ≤ 0.001) higher in the epithelial layer and the vaginal region. These findings are in accordance with our previous results and explain the differences observed in the HA content and differing HA fragment size at different stages of the estrous cycle.

Artificial insemination (AI) using frozen semen is a key method to enable rapid genetic improvement but its use in the sheep industry is currently limited by poor fertility. Laparoscropic AI is most effective but has not gained popularity due to cost and welfare considerations. Transcervical intrauterine AI (TCAI) may offer a practical alternative but the complex anatomy of the ovine cervix limits adequate penetration of the inseminating pipette. Hyaluronan (HA) is a glycoaminoglycan whose content in the cervix increases at oestrus and which may contribute to the degree of natural relaxation observed at this time. This study investigated the effect of intracervical application of HA on the depth of cervical penetration in sheep. Oestrus was synchronised on three occasions in 48 Welsh mountain ewes with progesterone sponges and PMSG. Each animal initially served as its own untreated control. Ewes were subsequently treated intracervically with 25 mg of: (2i) low molecular weight (MW) HA; (2ii) high MW HA or (2iii) both low and high MW HA (n = 16/group) at 52 h after sponge removal or with low MW HA at: (3i) 50 h; (3ii) 52 h or (3iii) both 50 h and 52 h. Depth of cervical penetration measured at 54 h was increased from 1.22 cm to 3.66 cm by treatment with low MW HA (P ≤ 0.001), with no differences between the number of treatments (1 or 2) or the time at which the HA was administered (50 or 52 h). High MW HA alone or together with low MW HA had no effect. In conclusion, intracervical application of low MW HA 52 h after sponge removal increases cervical penetration up to 3.4cm to allow TCAI in sheep.

This study investigated the effect of growth parameters and metabolic indices during the rearing period on the fertility of nulliparous Holstein-Friesian heifers managed on 17 UK dairy farms. Growth parameters (body weight [BW], heart girth, height, and crown-rump length) and metabolic indices (insulin-like growth factor-I [IGF-I], insulin, glucose, and urea) were measured at approximately 30, 180, and 450 d of age. Fertility data collected included age at first breeding (AFB), number of services per conception, pregnancy rate to first artificial insemination (AI), and age at first calving (AFC). Of the heifers initially bred (n=428), 4% failed to conceive. The mean pregnancy rate to first AI for heifers that conceived and calved without suffering reproductive loss (n=392) was 67%, and 6% required2 inseminations. The mean AFB and AFC was 473+/-5 d (range, 357 to 936 d) and 791+/-6 d (range, 636 to 1529 d), respectively. Increased BW, girth, and IGF-I concentration (at 30, 180, and 450 d) and increased skeletal growth (at 180 and 450 d) was associated with a reduced AFB and AFC (P0.05 to 0.001). Heifers calving at775 d had a mean BW gain of 0.82+/-0.01kg from 30 to 180 d. Increased glucose concentration at 180 d was associated with a reduced AFB (P0.01), but no associations were found between insulin and urea concentrations and any of the fertility traits recorded (P0.1). Suboptimal growth associated with an increased AFC could be alleviated by improved monitoring of replacement heifers during the rearing period.

Reproductively normal crossbred beef heifers were individually offered a diet of barley straw and concentrate supplemented with one of four levels of a fish oil (FO) enriched supplement. Following oestrous cycle synchronisation, blood samples were collected at appropriate intervals for the measurement of progesterone (P(4)), oestradiol (E(2)), fatty acids, insulin-like growth factor 1 (IGF-1) and metabolites. On days 15 and 16 of the cycle, oxytocin was administered intravenously and the prostaglandin F(2alpha) (PGF(2alpha)) response was measured as venous concentrations of 13,14-dihydro-15-keto PGF(2alpha) (PGFM). The heifers were slaughtered on days 17 or 18 of the oestrous cycle and endometrial tissue, rumen fluid and follicular fluid were collected for determination of fatty acid concentrations. In general there was no effect (P0.05) of diet on plasma P(4) or E(2) concentrations. Increasing FO supplementation increased CL diameter on day 7 post-oestrus (P0.0001) but had no effect on diameter on day of slaughter (P0.05). On day 15, PGFM concentration was greater on the highest level of FO supplementation compared to controls (P0.05), however, there were no differences between other diet comparisons (P0.05). There was no effect of diet on PGFM concentration on day 16 (P0.05). There was a strong positive relationship between plasma and uterine endometrial concentrations of both EPA (R(2)=0.86; P0.0001) and total n-3 PUFA (R(2)=0.77; P0.0001). IGF-1 concentrations increased on all diets and were greatest at the highest level of n-3 PUFA supplementation (P0.05).

A meta-analysis was performed to consolidate the results of studies which have evaluated the effects of Vitamin E supplementation during the dry period on the risk of retained foetal membranes (RFM) in the dairy cow. Twenty studies demonstrated a beneficial response to Vitamin E whilst 21 found no benefit and 3 reported an increase in the incidence of RFM in treated cows. The odds ratios (OR) of the available studies exhibited significant heterogeneity, so multivariable logistic regression analysis was performed to enable the identification of factors associated with the response to Vitamin E supplementation. Our multivariable analysis included parity and Vitamin E supplementation (control/treated) in the model, because all other factors were co-linear. Results indicated that Vitamin E supplementation led to a reduction in the incidence of RFM. A second multivariable analysis was undertaken on a subset of the data including only supplemented cows to determine the influence of supplementation factors on the risk of RFM. All factors were co-linear with each other, therefore, only type of Vitamin E supplementation was included in this analysis. The regression model demonstrated that administration of the synthetic Vitamin E alpha-tocopheryl acetate was associated with a lower risk of RFM than treatment with natural Vitamin E (alpha-tocopherol) (P=0.047, OR=0.49), whereas the difference between the synthetic Vitamin E alpha-tocopherol acetate and natural Vitamin E just failed to attain statistical significance (P=0.059, OR=0.53). Overall the analyses indicate that Vitamin E supplementation during the dry period is associated with a reduced risk of RFM, and that the synthetic forms of Vitamin E are more effective than the natural compound.

The peripartum period is of critical importance to subsequent health and fertility. Most cows enter a state of negative energy balance (NEB) associated with many metabolic changes which have carry over effects on the resumption and normality of estrous cyclicity and the success of subsequent inseminations. A dataset on 500 lactations explored the relationships between metabolic traits measured before and after calving with fertility. Stepwise multiple regression analysis showed that longer calving to conception intervals were associated with altered profiles of IGF-I, urea and body condition score. These relationships between metabolic profiles and fertility differed between first lactation cows (which are still growing but produce less milk) and mature animals. Early postpartum the liver undergoes extensive biochemical and morphological modifications to adapt to NEB, the uterus is extensively remodeled and must clear bacterial infections, and the ovary must resume ovulatory cycles. RNA isolated from liver and uterine tissues harvested 2 weeks postpartum from cows in mild (MNEB) and severe (SNEB) energy balance was used to screen the Affymetrix 23K bovine microarray. In liver, SNEB resulted in differential expression of key genes involved in lipid catabolism, gluconeogenesis, and the synthesis and stability of IGF-I. This was accompanied by reduced systemic concentrations of IGF-I which is likely to impact on ovarian function and early embryo development. Within endometrium, cows in SNEB showed histological evidence for higher levels of inflammation and the microarray analysis identified groups of differentially expressed genes involved in tissue remodeling and immune response. This may delay uterine repair after calving, likely contributing to the observed reduction in fertility.

Genetic selection has resulted in larger cows with high milk production potential but a tendency for poor fertility. In multiparous cows fetal development competes for nutrients with concurrent milk production. This study tested the hypotheses that (a) maternal age and milk yield during pregnancy alter calf birth size and (b) birth weight influences subsequent productivity and fertility. Concurrently born Holstein-Friesian heifers (n=65) with multiparous dams and three sires were monitored from birth to the end of their first lactation to assess effects of birth weight on growth, milk production and fertility. Calves were analyzed as three subgroups: low (L), average (A) and high (H) birth weight (BW) calves (n=21-22 per group). LBW calves were born 10 kg lighter than HBW calves and remained significantly lighter throughout the study. They were generally smaller in other measured indices (length, height, girth, ponderal index) between birth and 9 months and were more likely to have older dams (lactations 3-6) with higher peak yields (>42 kg/day). Milk production parameters were indistinguishable between the 3 birthweight groups and metabolic parameters (IGF-I, insulin, glucose) measured around first calving were unaffected. HBW offspring were more likely to have persistent corpora lutea following their first calving and other fertility parameters also tended to be worse. Sire influenced gestation length but not birth size. Sire heritability estimates showed that weight, IGF-I and insulin concentrations after first calving and fertility in the first lactation were all heritable. The results support the hypothesis that high milk production in the dam may predispose to birth of a smaller calf. Smaller birth size did not, however, have any subsequent adverse effects on productivity or fertility in the first lactation and sire was more influential at this stage.

Cows in severe negative energy balance after calving have reduced fertility, mediated by metabolic signals influencing the reproductive system. We hypothesised that transition diet could alter metabolic status after calving, and thus influence fertility. Multiparous dairy cows were assigned to four transition groups 6 weeks pre-calving and fed: (a) basal control diet (n = 10); (b) basal diet plus barley (STARCH, n = 10); (c) basal diet plus Soypass (high protein, HiPROT, n = 11); or (d) no transition management (NoTRANS, n = 9). All cows received the same lactational diet. Blood samples, body weights and condition scores (BCS) were collected weekly. Fertility parameters were monitored using milk progesterone profiles and were not affected by transition diet. Data from all cows were then combined and analysed according to the pattern of post-partum ovarian activity. Cows with low progesterone profiles had significantly lower insulin-like growth factor-I (IGF-I) and insulin concentrations accompanied by reduced dry matter intakes (DMIs), BCS and body weight. Cows with prolonged luteal activity (PLA) were older and tended to have lower IGF-I. Analysis based on the calving to conception interval revealed that cows which failed to conceive (9/40) also had reduced IGF-I, BCS and body weight. Fertility was, therefore, decreased in cows which were in poor metabolic status following calving. This was reflected in reduced circulating IGF-I concentrations and compromised both ovarian activity and conception. There was little effect of the transition diets on these parameters.