Potential Importance of Circulating anti-Müllerian Hormone as a Predictor of Superovulatory Response in Dairy Holstein Cows

Plasma anti-Müllerian hormone concentration (AMH) in cows is a useful endocrine marker for ovarian response to gonadotropin superstimulation. Here, we investigated the possible relationship between AMH concentration measured in plasma and superovulatory response of embryo donor dairy Holstein cow aiming to predict their response in earlier period before beginning of superovulatory treatment protocol. So, for this reason, 33 cows were undergo superovulatory gonadotropin treatment and blood samples were collected at three phases (P0 randomly before treatment, PE on day of estrus, PF on day of flushing for embryos collection). This study revealed that AMH concentration were significantly increased in the high responded cows in all 3 phases of study than the low responded ones. Also, the study reported 3 optimal cut-off points 57.6, 192.1 and 114.2pg/ml in the P0, PE and PF phases respectively which could be used for early prediction of cow superovulatory responses. In conclusion, circulating AMH concentration was strongly associated with superovulation response and its evaluation could be used to identify embryo donor cows with greater response to superovulation programs. Also, the optimal cut-off point 57.6pg/ml could be used randomly to select the best donors (Key wards: Superovulation, anti-Müllerian Hormone (AMH), dairy cows) Introduction Recent advances in bovine biotechnology, such as commercially available genomic testing, have allowed for the identification of animals with superior genetics. Genetic selection and reproductive efficiency are key factors for the success of the dairy and beef industries. In cattle, Multiple Ovulation and Embryo Transfer (MOET) programs have become a large international business. More accurate identification of cows with greater embryo production potentials could allow for more efficient production of in vitro and in vivo bovine embryos for cows with superior genetics (Rico et al., 2009). However, cost efficient propagation of these superior genetics has been hampered by high variability between animals in response to embryo production techniques such as superovulation (Souza et al., 2015). Despite improvements in superovulatory treatments, ovarian responsiveness to gonadotropins remains highly variable between individuals and difficult to predict. It is well established that the major source of variability is the status of ovarian follicles at the time of initiation of FSH treatment (Rico et al., 2009). Presently, some clinical studies thrown its light on anti-Müllerian Hormone (AMH) describing it as the best endocrine marker of ovarian follicular reserve, largely replacing other serum markers such as inhibin B, estradiol, basal FSH and LH (Fanchin et al.,2003; Broekmans et al., 2006; Toner and Seifer, 2013). AntiMϋllerian Hormone is also the best predictive marker of the ovarian response to stimulatory treatment as defined by the number of oocytes retrieved In Assisted Reproductive Technology (ART) (El-Gindy et al., 2008). Moreover, information is also accumulating in cattle proving that measurement of circulating AMH concentrations may be the most reliable method for predicting not only fertility potential and reproductive longevity (Baruseli et al., 2015; Jimenez-Krassel et al., 2015; Manal et al., VMJG Vol.63 (3) No. 27 -37 July 2017 ISSN1110-1423 28 2016) but also the relative number of morphologically healthy follicles and oocytes in ovaries (Ireland et al., 2008; Ireland et al., 2011; Monniaux et al., 2013; Batista et al., 2014). Also, during the bovine estrus cycle two to four sequential waves of terminal follicular growth occur, each producing a dominant follicle capable of ovulating, if luteal regression occurs (Fortune et al., 2001), and variations in concentrations of AMH during emergence and regression of follicular waves remain to be established (Rico et al., 2011) Anti-Müllerian Hormone (also known as Müllerian Inhibiting Substance, MIS) is a glycoprotein of 140-KDa that is member of transforming growth factor-β superfamily (TFGβ) of growth and differentiation factors, knowing as a gonadal hormone expressed only in gonads (Pepinskey et al., 1988). The sexually dimorphic regulation of AMH expression is a fruitful area of inquiry since it is early marker in mammals for the genetic switch that occurs when a bipotential gonads is instructed to differentiate into a testis in response to the testisdetermining factor, SRY (Sex – determining Region of the Y-chromosome) (Swain and Lovell,1999; Capel,2000). Müllerian Inhibiting Substance (MIS) has long been known for its signature developmental effect of causing regression of Müllerian duct, the anlagen of fallopian tubes, uteri, cervix and upper vagina, a requirement of normal male reproductive tract development. Whereas,T ,another hormone produced by fetal testis , is required for Wallfian duct differentiation into male internal reproductive tract structures (Teixeira and Donahoe, 1996). In the ovaries, AMH expression is restricted to a single cell type that is granulosa cells of growing follicles as recorded by previous studies in many species (in cows, Vigier et al., 1984; Monniaux et al., 2008, in sheep, Bezard et al., 1987, in human, Rajpert et al., 1999, Weenen et al., 2004). Anti-Müllerian Hormone is of key importance as it inhibits the recruitment of primordial follicles into the pool of growing follicles, and it decreases the responsiveness of growing follicles to Follicular Stimulating Hormone (FSH) (Durlinger et al., 2002). This pattern of expression makes AMH a reliable endocrine marker of the population of small antral gonadotropin responses follicles in the cows (Rico et al., 2009). Thus, the objective of this study was to investigate whether circulating AMH concentrations could be used as a predictor for superovulation response by a single measurement before beginning of superovulatory treatment in dairy Holstein cows. In addition, to detect an optimal cut-off point which could be used to select the best donors. Materials and Methods Animal Housing and Diets: Thirty-three multiparous dairy Holstein cows (3 to 7 years) old and their body weight ranged from 450 to 800 Kg.were used in this experiment which was conducted at a private farm at Alex-Cairo desert road. The experiment was done from November 2015 to January 2016. Experimental animals were assigned in an open yard and were provided feed and water ad libtum. Nutrient concentration met nutrient requirements of dairy cattle according to NRC (2001). They were fed concentrated ration (corn, soya bean meal and premix) in addition to afla afla and corn silage of high quality. The amount of ration was calculated according to the amount of milk production. Experimental Design and Superovulation Protocol: This experiment aimed to establish the relationship existing between circulating AMH concentration in plasma on one hand and ovulatory responses to a superovulatory treatment on the other hand. Table (1): Superovulation protocol (Rivera et al., 2011). VMJG Vol.63 (3) No. 27 -37 July 2017 ISSN1110-1423 29  Intravaginal progesterone (P4) implants (EaziBreed CIDR; containingn1.38g of P4, Pfizer Animal Health, New York, NY).  GnRH (Buserlin acetate, 1.0 μg/dose, Receptal; Intervet, International GmbH, Germany).  FSH (400mg, Folltropin-V; Bionich Life Sciences, Belleville, ON, Canada).  PGF2α (Estrumate, 250μg of cloprostenol sodium/dose; Shering-Plough Animal Health, Union,NJ). Cows were inseminated three times at 12hrs. interval by 2 straws of frozen semen (15x 10 6 sperm/straw) from Holstein sire with high genetic merit, proven outstanding field fertility (sire conception rate scores ≥2). CL Counting and Embryos Collection: Donor was contained in a crush, usually with head-tail restraint. Then a well-trained vet examined both ovaries for determining the total number of CL that may be corresponding to the total number of embryos may be collected. We used an epidural anesthetic for relaxation of the rectum and local effect on the tract to aid manipulation. The collection catheter is passed through the cervix and located in place usually in each of the uterine horns in succession, by manipulation with a hand insertion in the rectum (similar to manipulation of the cervix during AI). When the catheter is correctly located, the cuff is inflated and flushed medium passed through to collect the embryos. Then they were located and retrieved from the flushed fluid using a microscope, assessed for quality on appearance and stage of development, and then either prepared for transfer or frozen for storage. Blood Sampling: By jugular vein puncture, blood samples from each cow were received in clean and sterile tubes containing EDTA at three different phases: P0 representing the samples that were taken before superovulatory treatment (random). PE representing the samples that were taken after superovulatory treatment at time of estrus. PF representing the samples that were taken 7 days after estrus where counting of CL was done and flushing for embryos collection was taken place. Collected blood samples were immediately kept in icebox and later centrifuged at 3000rpm/15 min. for plasma separation. Then samples were kept frozen at -20 untill AMH assay were done. Plasma AMH Assay: Bovine antiMüllerian Hormone was assayed by ELISA technique using commercial diagnostic kit (Nova Tec, Immudiagnostica GmbH Waldstraβe 23 A6, D-63128 Dietzenbach, Germany). The assay had a sensitivity of 0.04pg/ml and coefficient of variation of 2.2٪. This assay had high sensitivity and excellent specificity for detection of bovine AMH. No significant cross-reactivity or interference between bovine AMH and analogues was observed. Statistical Analyses: Statistical analyses were performed using Sigma Plot version 13 (Systat Software Inc. San Jose, CA). The sensitivity and specificity of the test were calculated according to the formula that use the median values to determine the high (true positi