Relationship of Anti-Mullerian Hormone to Reproductive Traits in Katahdin Ewes Bred in Late Spring or Fall

Anti-Mullerian hormone (AMH) is an endocrine marker for fertility in many species. This study investigated possible correlations between serum AMH concentrations, mean number of lambs born (out-of-season in spring or fall), and estimated breeding values (EBVs) for number of lambs born (NLB), number of lambs weaned (NLW), maternal weaning weight (MWWT), weaning weight (WWT), and maternal index (determined by Sheep Genetics Australia). Blood was collected at breeding from Katahdin ewes between 0.7 and 7 years of age in 2015 (n = 163) for the analysis of serum concentrations of AMH. Anti-Mullerian hormone concentration was either expressed quantitatively or divided into quartiles (AMH Q1 through Q4, with Q1 the lowest and Q4 the highest, pg/ml). Data were analyzed by PROC CORR, GLM or chi-squared using SAS. Mean serum AMH was 182 ± 11 pg/ml and ranged from 0 to 1112 pg/ml. There was no correlation between serum AMH and EBVs for NLB, NLW, MWWT, WWT and the maternal index. Serum AMH concentration was similar in ewes in different age categories (P = 0.157). There was a correlation between EBV for NLB (0.29; P = 0.0002) and NLW (0.19; P = 0.013) with average number of lambs born. Ewes in the lowest AMH quartile (Q1) had a lower mean number of offspring born from spring breeding compared with ewes in other AMH quartiles (Q2, Q3, and Q4; P < 0.05). Further study is needed to determine the effectiveness of using serum AMH for selecting ewes for out-of-season (spring) breeding.


Introduction
Anti-Mullerian hormone (AMH) is a dimeric glycoprotein that belongs to a member of the transforming growth factor-β (TGF-β) family [1]. This hormone is produced by granulosa cells of pre-antral and small antral follicles of 1 -3 mm diameter [2] [3]. Anti-Mullerian hormone facilitates follicle recruitment and growth of young follicles [4]. However, when a follicle increases in size, AMH limits the number of follicles developing from the gonadotropin responsive to the gonadotropin dependent stage [5]. Although AMH limits the number of ovulatory follicles, studies have positively correlated plasma AMH with fertility in sheep [6] [7]. Studies with cattle have shown that AMH concentration remains constant in an individual throughout the estrous cycle and during active reproductive age [3] [8] [9] [10].
It is well understood that individual ewes vary in reproductive performance, which is the basis for estimated breeding values (EBVs). Estimated breeding values (EBVs) are the mathematical predictions of the genetic merit of an animal for specific traits including reproduction. These values are calculated based on an individual animal's performance for a trait compared with animals within a contemporary group, and animals in other genetically connected flocks. Estimated breeding values are useful to select for reproductive or maternal traits used in breeding or commercial Katahdins, including prolificacy or number of lambs born (NLB) or weaned (NLW), or maternal weaning weight (MWWT), weaning weight (WWT), and maternal index (http://www.nsip.org/). As lambs, EBVs are an estimate of the parent's genetic potential until sufficient data is collected on an individual and siblings. Similarly, the ability to conceive out-of-season also varies among ewes [11], but to date there is no way to predict whether an animal is likely to breed out-of-season. It is useful to predict reproductive performance of ewes early in life to minimize costs to rear the most efficient ewes.
Studies are lacking on relationship between serum concentrations of AMH and maternal EBVs in ewes, or out-of-season breeding success. Therefore, the objective of this study was to examine the relationship between serum AMH, mean number of lambs born (in spring, out-of-season or fall), and EBV for NLB, NLW, MWWT, WWT, and the maternal index.

Animal Management
Katahdin ewes were raised at USDA, ARS, DBSFRC in Booneville, AR. A total of 163 ewes ranging from 0.7 to 7 years of age were categorized into 5 age groups: less than 1 (n = 17), 1 to 2 (n = 57), 2 to 3 (n = 23), 3 to 4 (n = 34), and more than 4 years of age (n = 32). Ewes were maintained on predominantly tall fescue (Schedonorus arundinacea) and/or bermudagrass (Cynodon dactylon) overseeded with winter annuals [Wheat (Triticum aestivum L.) and rye (Lolium multiforum L.)] and were supplemented with corn/soybean meal (17% CP, 82% TDN on DM basis). Rams passed breeding soundness evaluation before breeding, and ewes were over 6 months of age and weighed at least 35 kg. Ewes were exposed to rams for approximately 30 days either in fall (August/September) or spring (April/May) at a ram to ewe ratio of 1:20 to 1:30. Body weight of ewes was recorded on first day of ram exposure, and offspring were weighed at birth and approximately 60 days of age.
In some years ewes that would have been eligible to breed in fall were held over for spring to increase number of ewes available at that time.

Ovine AMH ELISA Immunoassay
The equine and ovine AMH ELISA kit used was a quantitative three-step sandwich type immunoassay (AL-115, AnshLabs, Webster, TX

Statistical Analysis
For analysis, pregnancy rate is defined as the proportion of ewes that were pregnant approximately 28 days after ram was removed from breeding groups. One ewe did not lamb after being diagnosed as pregnant, but all other ewes deter-

Results
Intra-assay variability of AMH assays ranged from 6.6% to 8.3%. Inter-assay variability between standards was 4.3%. Least square mean serum concentration of AMH was 182 ± 11 pg/ml and ranged from 0 to 1112 pg/ml.

Correlation of EBVs with AMH Concentrations and Average Number of Lambs Born
Serum

Relationship between AMH Quartiles and Mean Number of Lambs Born from Spring (Out-of-Season) or Fall Breeding
There was no correlation between AMH quartiles with mean number of lambs born from fall breeding (P > 0.05; Table 1). However, there was a significant correlation between mean number of lambs born from spring breeding with AMH quartiles (P < 0.05). Ewes in lowest quartile (Q1) had lower number of lambs born from spring breeding than ewes in other quartiles (Q2, Q3, Q4; P < 0.05).

Pregnancy and Lambing Rate of Exposed Ewes between Breeding Seasons
Overall pregnancy rate was higher (P < 0.01) in fall bred ewes (79.0%) compared with spring bred (48.0%). Mean number of lambs born from fall breeding (1.44 ± 0.05) was higher (P < 0.01) compared with mean number of lambs born from spring breeding (0.78 ± 0.05) per ewes used for breeding.

Lambing Rate per Parity
Lambing rate was highest in third parity ewes and lowest in first parity ewes (P < 0.05). Mean numbers of lambs born at first, second, third, fourth, fifth, and sixth

Discussion
The mathematical calculations of EBVs are complex, taking into account of phenotypic (body weights, type of birth) and genetic (using data from relatives entered into NSIP) parameters. The EBV is the best estimate of the true genetic merit of any individual for a specific genetic trait measured (http://www.nsip.org/).
The more data or genetic connections within the breed, the greater the accuracy of the EBV [12]. In the current study, there was no correlation between serum Ovarian follicular population, an indicator for fertility, was positively correlated with plasma AMH concentration in young lambs [6] [7]. A positive correlation between plasma AMH concentrations during the prepubertal period and occurrence of ovulation in response to eCG was also reported [6]. A higher number of embryos were found in synchronized ewes with higher plasma AMH concentrations [14]. Ewes with a high plasma AMH (>97 pg/ml) had a higher number of lambs born per ewe per year until 3.5 years of age compared with low (<97 pg/ml) plasma AMH concentrations measured at 3.5 months of age [7].  [15]. In our previous study, we reported AMH concentration of heifers at breeding, with the lowest quartiles (Q1) had lower AI pregnancy rate and conceived at a later estrus cycle in breeding compared with heifers in higher quartiles [15].
Out-of-season breeding during long days is beneficial to the sheep industry for several reasons. First, breeding in the spring results in late-gestation and lactation coinciding with rise in high quality cool-season forage in the southeastern US [16]. Second, weather in early fall is ideal for pasture lambing, reducing chances of weather related deaths in young [17]. Third, lambs can be marketed in spring when demand is high and the lamb supply is low [18]. In much of the US, pregnancy and lambing rates are higher during fall breeding compared with spring, and Katahdins in the Southeastern US are no exception [11]. Katahdin ewe lambs or yearlings have more pregnancy losses, and lower lamb birth weights when bred during spring compared with fall [11]. In order to increase spring breeding success, it will be useful to identify ewes that are capable of out-of-season breeding. In the current study, ewes in lowest AMH hormone quartiles had lower number of lambs born from spring (out-of-season) breeding, thus further study can be done to select ewes that can breed out-of-season by using AMH endocrine marker.
Anti-Mullerian hormone is high during the fetal stage compared with postnatal concentrations [2]. Similarly, plasma AMH concentrations in 40 d old ewes are higher than 3.6-month old ewes [6] [19]. Lahoz found a vast difference between 1 and 5-month-old ewes, decreasing with age [20]. Serum AMH concentrations of ewes after puberty, at different reproductive ages has not been reported. In the current study, serum AMH concentrations was similar in different age groups (less than one, 1 -2, 2 -3, 3 -4, and more than 4 year old) ewes. Age did not influence serum AMH concentrations as previously reported in cattle [8] [9].

Conclusion
In conclusion, serum AMH concentration was similar among age groups. Ewes in the lowest quartile (Q1) had lower number of offspring born from spring breeding compared with ewes in other AMH quartiles (Q2, Q3, and Q4). However, AMH was not useful in predicting fertility or maternal traits based on EBV of the ewes. Further research can be done to select ewes that are capable of out-of-season breeding using endocrine markers.