Evaluation of out-of-season estrus induction protocols in progesterone-primed mix-breed dairy goats using ecg, GnRH and E2

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1 B-689 [1-5] Indian J. Anim. Res., Print ISSN: / Online ISSN: AGRICULTURAL RESEARCH COMMUNICATION CENTRE Evaluation of out-of-season estrus induction protocols in progesterone-primed mix-breed dairy goats using ecg, GnRH and E2 Evaristo Carrillo 1, César A. Meza-Herrera 2, Juan R. Luna-Orozco 3, Ramón A. Delgado-Gonzales 4, Leticia R. Gaytan Aleman 4, Oscar Ángel-Garcia 4, Francisco G. Véliz 4 and Viridiana Contreras-Villarreal 4 * Universidad Autónoma Agraria Antonio Narro, Departamento de Ciencias Médico Veterinarias, Torreón, Coahuila, México Received: Accepted: DOI: /ijar.v0iOF.9116 ABSTRACT Different out-of-season estrus induction protocols were evaluated using a single progesterone (P4) priming 24h prior the use of ecg, GnRH or E2. Adult anestrus mix-breed dairy goats (n=32) were P4-primed (20 mg i.m.) and randomly assigned (n=8) to: a). E2: 1mg i.m. estradiol+0.2ml cloprostenol 24h later, b). E2+GnRH: E2+2.5ml GnRH 24h later, c). E2+eCG, E2+100 IU i.m. ecg 24h later, and d). ecg, 250IU i.m. ecg. Response variables considered the percentages of estrus (ES%, detected by sexually active males), ovulation (OV%), and pregnancy (PR%, diagnosed 45d after estrus detection). All treatments achieved high ES% (>87%) and OV% (>50%), yet, E2+GnRH reached 88% of OV% and PR%. E2 alone had the lowest PR% (p<0.05; 12.5%) and the highest interval from estrus to ovulation. The largest PR% (p<0.05; 81.5%, average) were observed in E2+GnRH and E2+eCG. Results indicate the feasibility of a simple synchronization protocol based in a single i.m. progesterone-priming plus E2+GnRH. Key words: Anestrous season, Estrus percentage, Exogenous hormones, Ovulation induction, Ovulation percentage. INTRODUCTION In some places of the arid regions of northern Mexico, specifically in the Comarca Lagunera (26 N), goats breeding season ranges from late July to early February, with an anestrus season from late February to early June (Luna- Orozco et al. 2012). As a consequence, there are wide seasonal fluctuations in both meat and milk production and supply, a situation that promotes a huge oscillation in the payments received by goat producers (Gonzales-Bulnes. 2011) which negatively affects farmer s economy. The design of out-of-season protocols to promote ovulation are commonly based on controlled internal drug release (CIDR) or intravaginal polyurethane sponges impregnated with progesterone (P4) or a synthetic analogue (progestogens) plus equine chorionic gonadotropin (ecg) and estrogenic pharmacologically active substances (Simoes 2015). According to Alvarado-Espino et al., (2016), the combined use of P4-priming plus a 100-IU hcg injection was an effective protocol for inducing estrus in non-cycling Alpine goats during the anestrus-to-estrus transition period. In addition, a single injection of P4 + ecg was a pessary-free protocol -either CIDR or intravaginal sponge-, that successfully induced and synchronized ovulation, generating high pregnancy rates in goats (Contreras-Villarreal et al. 2016). Despite ecg being a commonly used glycoprotein, there is an amplified immune response when such treatment is carried-out in a recurrent fashion (Drion et al. 2001). Moreover, if sustainability of most animal production systems is to be achieved, such animal systems must redesign out-ofseason ovulation induction protocols considering appropriate standards of animal welfare while decreasing the use and (or) dose of external hormones, since their use has been strictly regulated in several countries (Simoes 2015). The working hypothesis of this study is that a simple estrus induction protocol based in a single i.m. P4-priming plus the use of E2 + GnRH, is effective in synchronizing estrus and ovulation, generating important reproductive out-of-season outcomes in adult anestrus goats under rangeland conditions. MATERIALS AND METHODS General: All methods and management of the experimental units used in this study were in strict accordance with accepted guidelines for ethical use, care and welfare of animals in research at international (FASS, 2010), national (NAM, 2002) and institutional levels, with approval reference number UAAAN/UL/ Environmental conditions, animal management and experimental design: The experiment was carried out in the semi-arid lands of northern Mexico (Comarca Lagunera, 26 *Corresponding author s dra.viridianac@icloud.com 1 Instituto Tecnologico de Torreón, Torreón, Coahuila, México 2 Universidad Autónoma Chapingo, Unidad Regional Universitaria de Zonas Áridas, Bermejillo, Durango, México, Centro de Bachillerato Tecnológico Agropecuario, Nº 1, Torreón, Coahuila, México 4 Universidad Autónoma Agraria Antonio Narro, Departamento de Ciencias Médico Veterinarias, Torreón, Coahuila, México

2 2 INDIAN JOURNAL OF ANIMAL RESEARCH N, 104 W; altitude 1,140 m), from May to June. The area is dominated by a microphyll desert scrub, mean annual temperature is 27 C while average annual precipitation is 230 mm, with 70% falling from June to October. The average day length in June and December is 13 h, 41 min and 10 h, 49 min, respectively. At the beginning of the natural anestrous season, multiparous mix-breed dairy goats from a commercial herd kept under rangeland-extensive conditions were evaluated by means of transrectal ultrasonography scanning (Aloka SSD500 linear array; Overseas Monitor Corp. Ltd., Richmond, BC, Canada) by a trained operator to confirm the anovulatory state of the goats. All goats in this study were not pregnant, had kidded the previous year and were isolated from bucks four months prior the onset of the experimental protocol. Goats (n=32) were primed with a single i.m. P4- dose of 20 mg (Progesterona Cheminova, Mexico, D.F., Mexico) and then randomly assigned to one of four experimental treatments (n=8 per group). The experimental treatments were performed 24 h after P4-priming. The experimental treatments considered: 1). E2, treated with 1 mg i.m. estradiol (cyclopentyl propionate estradiol, Parfarm, Mexico, D.F., Mexico) and 0.2 ml i.m. cloprostenol (Lab. Sanfer, Mexico, D.F.) 24 h after E2 administration. 2). E2+GnRH, E2 plus, 2.5 ml i.m. GnRH (Lab. Sanfer, Mexico, D.F., Mexico) 24 h after E2 administration. 3). E2+eCG, E2 plus, 100 IU i.m. ecg (Parafarm, Mexico, D.F., Mexico). 4. ecg, treated with 250 IU i.m. ecg 24 h after P4 administration. At the beginning of the trial, goats average live weight was 54.5 ± 0.07 kg; the animals were assigned to different pens according to treatment and all the experimental groups were located away from contact with males and away from each other. During this period, goats were fed with alfalfa hay ad libitum, had access to mineral blocks and 200 g of commercial concentrate (14% CP) per day per animal. Characterization of the behavioral estrus activity: Estrus was detected with the use of intact crossbred bucks induced to sexual activity by the administration of testosterone (25 mg i.m. every 3d x 3 weeks, Lab Brovel, DF, Mexico), which were in contact with does every 8 h (0600, 1400 and 2200 h) during a 15 min period. Females were considered to be in estrus when they showed typical estrus behavior such as tail movements, peeing when buck approached them and allowed to be mounted. Estrus behavior detections started on day 1; hormonal treatments were applied around 0600 h and the first estrus behavior evaluation was performed the same day at 2200 h. Estrus was monitored during the next 168 h posttreatment; thereafter, goats were exposed and bred by sexually active bucks. Time from treatment to onset of estrus behavior was measured. Characterization of ovarian follicular dynamics: Does underwent ultrasonographic evaluation with a transrectal transducer (Aloka SSD500 linear array; Overseas Monitor Corp. Ltd., Richmond, BC, Canada), in order to detect and measure ovarian structures. A single experimented operator conducted the ultrasonographic scanning with does in standing position. The transducer was inserted into the rectum until an image of ovaries was observed on the monitor. Then the transducer was rotated until both ovaries were scanned. Follicles were measured using the measuring function available from the ultrasound machine (diameter in mm) and their locations on the ovaries were recorded daily from 7-d pre-treatments up to 7-d post-hormonal treatments. Maximal crosssectional area of each corpus luteum was registered 10 d after ovulation. The corpus luteum was identified on gray scale as hypoechoic area within an ovary. The size of this structure was calculated as the average of transverse, anteroposterior and sagittal diameters. Ovulation was detected by measuring the follicle with the maximal diameter and observing the morphologic changes within the ovary from follicular tissue to the formation of a corpus luteum. Time from onset of estrus to the time of ovulation was also measured. Pregnancy was evaluated with ultrasonography scanning 45 d after behavioral estrus was detected; thereafter, kidding percentage was recorded. Statistical analyses: The percentages of goats in estrus, ovulating and pregnant were analyzed as binomial data with the LOGIT function of the PROC GENMOD procedure of SAS (SAS Inst. Inc., Cary, NC, USA). The model statement contained the effect of hormonal treatment. For dimensions of ovarian structures, data were analyzed by the MIXED procedure of SAS using a model that included the hormonal treatment applied to does. Estrus length, time from treatment to estrus, treatment to ovulation, estrus to ovulation and follicular dynamics were analyzed with a Student T test. P-values less than 0.05 were declared to be significant. RESULTS AND DISCUSSION Results of this research demonstrated that a single primer intramuscular injection of progesterone in addition to ecg or E2 + either ecg or GnRH are highly efficient protocols to induce and synchronize estrus activity of previously anestrous mixed-breed dairy goats kept under rangeland-extensive conditions in northern Mexico (26 N). Table 1 shows estrus, ovulation and pregnancy rates and time from treatment to estrus (h), estrus to ovulation (h) and estrus length (h). All treatments were able to induce and synchronize estrus, observing an estrus response of >85%. There was a high ovulatory response (>85%) for E2 + GnRH, E2 + ecg, and ecg. These groups also had a high pregnancy rate (>81%). Estrus length was shorter for the ecg-treatment (29 h) than other treatments. Also, interval from estrus to ovulation was shorter for ecg group, meanwhile, the longest was for E2. The E2-group depicted the worst reproductive outcomes; the longest behavioral estrus (60±3 h), the lowest

3 Vol. Issue, () Table 1: Estrus, ovulation, pregnancy rates and estrus length (time from treatment to estrus, h) in mixed-breed dairy goats during the natural anestrus season and treated with a single dose of P4 plus either E2, GnRH, and ecg, kept under rangeland- extensive conditions in northern Mexico (26 N) Variables Treatment Group E 2 E 2 +GnRH E 2 +ecg ecg Estrus (n&%) 8/8 (100) a 8/8 (100) a 8/8 (100) a 7/8 (87.5) a Ovulation (n&%) 4/8 (50%) a 7/8 (88%) a 7/8 (88%) a 5/8 (62.5) a Pregnancy (n&%) 1/8 (12.5) a 7/8 (88%) b 6/8 (75%) b 5/8 (63%) a,b Treatment to estrus (h) a a a a Estrus to ovulation (h) a a,b a,b b Estrus length (h) a a,b a,b b a,b Means with different superscripts in the same column differ (P<0.05). ovulation rate (50%) and the lowest pregnancy rate (12.5%); such findings are in line with previous findings (Menchaca and Rubianes 2007). An explanation for this is that administration of exogenous estradiol mimics the actions of estradiol secreted by a dominant follicle and while it is successful for behavioral estrus to take place, its levels were not enough to increase the required GnRH secretion in order to induce the pre-ovulatory LH surge needed for ovulation (Fatet et al. 2011). This endocrine imbalance could have caused anovulation of the dominant follicles (Sato et al. 2011; Tanaka et al. 2007). Follicular dynamics across treatments is presented in Table 2. The amount of small, medium and large follicles at day 0 were the same for all treatments, yet, on day 2, the groups E2 and E2+GnRH had more small follicles while E2 + ecg and ecg treatments had more large follicles. These large follicles could have been persisting follicles that failed to ovulate. Even though Tanaka et al. (2007) found that the exogenous treatment of progesterone and estradiol can artificially induce follicular cyst in cows, it seems that they do not induce ovarian cysts in cycling goats. Goats in the E2- group showed signs of continuous heat, which is a sign of follicular cysts (Matthews 1999) with a mean time from estrus to ovulation of 75 ± 5.7 h. The last was significantly longer than that shown by the other experimental groups, E2+GnRH and E2+eCG (48 ± 5.7 h and 46 ± 4.7 h, respectively) while the shortest was depicted by the ecg group (27 ± 4.5 h) (Table 1). This longer latency from estrus to ovulation enhances the hypothesis that most of these large follicles Table 2: Follicular dynamics on day 0 and day 2 of treatment in mixed-breed diary goats treated with a single dose of P 4 plus either, E2, E2 and GnRH, E2 and ecg or ecg kept under rangeland- extensive condition in northern Mexico. Day 0 Day 2 Small (3-4.4mm) Medium ( mm) Large (>5.5mm) Size ovulatory follicle (mm) Small (3-4.4mm) Medium ( mm) Follicle size Large at ovulation (>5.5mm) (mm) Ovulatory rate (n) E a a a a a a a E2+GnRH a a a a,b a b b E2+eCG a a a c a b b ecg a a a b a b b

4 4 INDIAN JOURNAL OF ANIMAL RESEARCH probably were follicular cysts. This would further explain the longer behavioral estrus showed by these goats, as these follicles would be producing estradiol for a longer period of time because of their failure to ovulate. ºThe average size of the ovulatory follicle at the time of ovulation was similar for all groups (p>0.05). The E2 + GnRH group had a high ovulation percentage of 88%, equal to that achieved by E2 + ecg, while a pregnancy rate of 88% similar to that of the E2 + ecg group (75%). Both treatments had a greater follicular growth than E2 with an average of large follicles of 2.5 ± 0.7 for E2 + GnRH and 3.1 ± 1.08 for E2 + ecg. These treatments also promoted a greater estrus synchrony suggesting that they promoted a homogenous follicular growth. Rubianes and Menchaca (2003) stated that if the pool of follicles shows a large heterogeneity, the response is characterized by an asynchronous follicular growth and luteinization, and this could be related to the mentioned inadequate responses. Ovulatory rate was 1.75 for E2+GnRH and ecg while groups E2 and E2 + ecg had an average of 1.2. Previous studies have demonstrated that administration of estradiol was unsuccessful in promoting optimum reproductive outcomes, with a decreased fertility rate (Menchaca and Rubianes 2004). In our study, E2 treatment had a greater variability on the estrus synchrony with does showing the first sign of behavioral estrus within 48 h after treatment (range h) compared to the 24 h achieved by the E2 + GnRH (range h) and E2 + ecg (range h), yet, no statistical differences were observed regarding the mean interval from treatment to the onset of estrus. This is of importance if an AI program is to be performed, as 100% of animals showed estrus within a 24 h period and a high percentage ovulated between h. Motlomelo et al. (2002) found that the overall mean interval to onset of estrus following progestogen (MAP, FGA or CIDR) withdrawal was 30.1±5.5 h. Our results show an overall mean interval to onset of estrus of 34.5±4.8 h. The normal duration of estrus behavior varies from 24 h to 48 h depending on age, individuals, breed, season and the presence of a male (Fatet et al. 2011). These parameters are similar to those found with E2 + GnRH (43 ± 4.4 h), while the ecg group had a short estrus that only lasted for 29 ± 0.7 h. Equine chorionic gonadotropin has a LH- and FSHlike activity on the theca and granulose cells of the follicle and is dose-dependent (Rensis and Lopez-Gatius 2014). The higher dose used in this group could explain the shortened time from estrus to ovulation. To conclude, results of our study indicate the feasibility of a simple synchronization protocol based in a single intramuscular progesterone priming injection plus the use of E2 + GnRH, obtaining important reproductive out-of-season outcomes in goats. Estrus induction, follicular growth, ovulation and pregnancy were similar with the use of the E2+GnRH and E2+eCG treatments. Furthermore, these treatments increased reproductive outcomes besides to the advantage of abolish the adverse effect of ecg administration, since recurrent treatments promote the formation of ecg antibodies. Results could be significant from a physiological, reproductive and productive stand point to the goat industry. REFERENCES Alvarado-Espino A.S., Meza-Herrera, C.A. Carrillo, E., Gonzalez-Alvarez, V.H., Guillen-Muñoz, et al. (2016). Reproductive outcomes of Alpine goats primed with progesterone and treated with human chorionic gonadotropin during the anestrus-to-estrus transi tion season. 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