Effect of two routes of administration of human chorionic gonadotropin upon oestrus induction and reproductive outcomes in adult acyclic mix-breed goats

ABSTRACT The effectiveness of two routes of administration of human chorionic gonadotropin (hCG) upon oestrus induction in anoestrus mix-breed goats was evaluated. Goats [n = 27, 39.6 ± 2.18 kg live weight and body condition score of 1.71 ± 0.02 units (scale 1–5)] were primed with 20 mg P4-intravulvosubmucosal (IVSM) 24 h prior to hCG administration. Thereafter, goats were randomly allocated to three groups to receive 100 IU hCG (0.1 mL) intramuscular (IM, n = 9), or IVSM (n = 9), and saline (0.5 mL) IM injection (CONT, n = 9). Ovarian follicular or luteal tissue was monitored by transrectal ultrasonographic scanning 15 days pre- and 5 days post-treatment. Besides, goats were daily monitored (0800 and 1800 h)  × 15 days to detect oestrus signs with the use of aproned sexually active bucks, treated with testosterone; 12 h after the onset of oestrus, goats were mated. While the CONT-goats never depicted neither oestrus nor ovulation (P < .05), the ovarian response from the IM and IVSM groups was 89% vs 68%, respectively (P > .05). Oestrus response (83.5%), pregnancy (60.6%) and litter size (1.55 ± 0.2) did not differ (P > .05) between the IM and IVSM groups. A single application of 100 IU hCG, irrespective of administration route, induced sexual activity in acyclic goats, generating key out-of-season reproductive outcomes.


Introduction
While reproductive technologies represent a unique opportunity for goat producers to improve the breeding value of their flocks, several efforts have been made to develop protocols to induce and synchronize oestrus and ovulation during both the breeding and non-breeding seasons (Simoes 2015). Improvements in the design of oestrus induction protocols have allowed to expand the out-of-season use of artificial insemination, inseminating goats at specific times, while achieving pregnancy rates comparable to those obtained with the use of visual detection of oestrus (Menchaca & Rubianes 2004;Simoes 2015). Common protocols to induce oestrus in acyclic goats include the combined or single use of gonadotropins, gonadotropin-releasing hormone (GnRH), progestins and prostaglandins (Esteves et al. 2013;Contreras-Villarreal et al. 2016;Sen & Onder 2016).
Nonetheless, in marginal-grazing production systems, the use of expansive reproductive technologies is not attractive to most producers because of the high costs of different drugs required to control the oestrus cycle (Gonzalez-Bulnes et al. 2011). Hence, efforts to simplify oestrus induction protocols with the concomitant cost reduction are essential in order for goat-keepers to use cheap, simple and effective protocols for out-of-season oestrus induction. One of the strategies to accomplish that is the elimination of progestins and prostaglandins from such protocols (Menchaca & Rubianes 2004). In acyclic goats, the use of human chorionic gonadotropin (hCG) alone could be an effective strategy to induce reproductive activity. Besides, although follicular development can be observed during the anoestrus period, such growth is quite limited, hampering the ovulatory stage (Sarath et al. 2012). Recently, Alvarado-Espino et al. (2016) reported that a single injection of 100 IU of hCG was able to induce oestrus response and ovulation in Alpine goats during seasonal anoestrus. Besides, despite the specific anastomoses involving the vulva, vagina, uterus and ovaries, it is still elusive if such angioarchitectural scenario may allow a more efficient action of drugs injected throughout the intravulvosubmucosal (IVSM) than the intramuscular (IM) route (Meira et al. 2006). This trial aimed to evaluate the use of a single shot of hCG administered by two different routes, either IM or IVSM, upon oestrus induction and some other reproductive outcomes in acyclic mix-breed goats.

Location and animal management
The present study was conducted in a commercial herd managed under extensive conditions during April-May, the natural non-breeding season for goats in Northern Mexico (25°||N, 1,140 m altitude) (Mellado et al. 2014). While the annual average for temperature is 23.5°C (range −2 to 43°C ), the annual relative humidity ranges between 28% and 81%. The annual rainfall in this area is 230 mm, and the day length is 13 h, 41 min at the summer solstice and 10 h, 9 min at the winter solstice. Goats grazed on open highly degraded rangeland typical of the Chihuahuan desert year round, driven by a herdsman (8 h per day; 1000-1800 h). Animals were penned near the household at night without access to feed and water. Goats kidded between September and December. Under the traditional management of these marginal production systems, goats receive neither food supplements nor minerals throughout the year, and were not treated against internal and external parasites, neither received any preventive vaccine management. To have a better control of the experimental units during the trial, goats were housed in three 4 × 4 m roofed pens and were offered alfalfa hay ad libitum plus 200 g concentrate with 14% crude protein content.

Confirmation of anoestrus status and experimental procedures
Pluriparous non-cycling mix-breed goats (Native × Alpine-Saanen dairy breeds) were used in the study. Averages for live weight (LW) and body condition score (BCS) were 39.6 ± 2.18 and 1.71 ± 0.2 kg. BCS was determined by tactile appraisal of fat in the sternum and lumbar vertebrae; scale 1-5 (Rusell et al. 1969). In order to confirm anovulatory status, prior to the onset of the experimental treatments, 30 goats underwent transrectal ultrasonographic scanning conducted by a single experimented operator, on days −15, −10 and −5 regarding the onset of the experimental treatments, using an Aloka 500 with a 7.5-MHz human prostate transducer (linear array; Corometrics Medical Systems, Inc., Wallingford, CT). During the ultrasonographic scanning, does were placed in the standing position and faeces were removed at the moment of the scanning. A coating of carboxymethylcellulose was applied to the transducer as a coupling medium. Once the uterine horns were clearly located, the transducer was rotated 90°clockwise and 180°counterclockwise across the reproductive tract until both ovaries were scanned in order to assess the functional status of the ovary; goats with a corpus luteum (n = 3) were discarded from the study.
Twelve hours after hCG administration, entire aproned bucks of proven libido were introduced to each experimental group in a proportion of 1:9. The does from all groups were exposed to the bucks at the same time, two times per day (0600 and 1800 h × 15 min) (Angel-Garcia et al. 2015). Does were considered in oestrus when they were mounted by the aproned bucks (Chemineau et al. 1992). Then, 12 h after, the confirmed-oestrus goats were exposed to one sexually active buck within treatment, with previous sexual experience and proven libido and fertility, treated with testosterone (testosterone cypionate im, 25 mg, every 3 days × 3 weeks prior to the goat's hormonal treatments; Luna-Orozco et al. 2012). Both oestrus detection and the experimental breeding were performed during a 15-day post-treatment period.

Ultrasonographic measurements of ovarian structures
Upon oestrus onset and once bred by the males, the goat's ovarian follicular activity was monitored up to day + 5 throughout daily transrectal ultrasonographic examinations. Ovarian images were obtained with a B-mode scanner (Aloka SSD 500, Overseas Monitor Corp. Ltd, Richmond, BC) equipped with a 7.5-MHz transducer (1.6 cm, diameter, 35 cm, length). The transducer was manipulated externally, with the doe in the standing position on a raised wooden platform and placed in a narrow chute. The image on the scanner was frozen at 1.5×; at this magnification, ovarian follicles around 1 mm were detected; but in order to reduce error, only diameter and position in both ovaries of those follicles >3 mm in diameter were recorded. Follicles of both the ovaries were counted, measured and classified as small (3-4 mm), medium (4.5-5.4 mm) and large (>5.5 mm). Diameter and position of corpora lutea were also recorded. Pregnancy was detected at 45 days post-breeding throughout transrectal ultrasound (Aloka SSD 500, 7.5 MHz linear transducer).
Response variables consider oestrus (%), ovulation (%), ovulation rate, dominant follicle diameter (>5.5 mm), interval of onset of oestrus-to-ovulation (h), oestrus length (h), pregnancy (%) and litter size. The does were considered in oestrus when they were mounted by the teaser bucks. Oestrus duration was defined as the time between the first and the last accepted mount, within the same oestrus period. Ovulation rate was determined by the disappearance of the dominant follicle view in the prior ultrasound examination and confirmed by the presence of corpora lutea on the surface of both ovaries of all goats. Additional variables recorded were pregnancy rate at day 45 post-oestrus, the interval from the start of treatment to oestrus and prolificacy, which consider the number of kids born per kidding doe.

Statistical analyses
The continuous variables time interval from treatment to oestrus onset, and follicle diameter, considered a general linear model with treatment as the main effect (PROC GLM, SAS Inst. Inc., Cary, NC). Mean values were compared according to a cut-off point of 0.05. No interactions were tested. Data on oestrus response, ovulation rate, pregnancy rate and kidding rate were analysed by categorical procedures using the PROC frequency procedure and genralized linear model procedures of SAS with the logit link function. The Wilcoxon Rank Sum test (the non parametric on way Wilcox Rank Sum test of SAS) was used to analyse prolificacy. Statistical differences among treatments were considered to be significant at P < .05.

Results and discussion
None of the goats in the control group presented oestrus, ovulation or growth of ovarian structures (Table 1), so reproductive variables were only compared between the hCG-treated groups. Certainly, while the CONT-goats never depicted neither oestrus nor ovulation (0%) (P < .05), the oestrus and ovarian response from the IM and IVSM groups was similar (89% vs 78% and 89% vs 68%, respectively (P > .05)). Results of our study confirm such hypothesis that the combination of P4-primed plus hCG in the present study was successful in inducing oestrus, regardless of the administration route, with quite notable reproductive outcomes, suggesting that this simple protocol, regardless of the administration route, not only activated the ovarian steroidogenesis pathway by the dominant follicles to induce oestrus behaviour, but also induced a preovulatory gonadotropin surge, which, at the end, lead to ovulation and luteinization.
The oestrus behaviour percentage observed in the present study is in agreement with that reported in other trials using a combination of fluorogestone acetate, prostaglandin F2ɑ (PGF2ɑ), equine chorionic gonadotropin (eCG) or follicle stimulating hormone (FSH) (Greyling & Van der Nest 2000;Fonseca et al. 2005a;Bukar et al. 2012), or more simple protocols (López-Sebastian et al. 2007). The proportions of does showing oestrus, during a 108-h period after the application of hCG, are shown in Figure 1. The interval from P4-primming to the beginning of oestrus was similar between groups of goats receiving the hCG treatment (IM, 63 ± 5.3 and IVSM, 54.9 ± 4.3 h; P > .05). This interval, ∼60 h, is close to other reports using medroxyprogesterone acetate for 6 and 9 days, eCG and PGF 2α (Greyling & Van Niekerk 1990;Fonseca & Torres 2005;Fonseca et al. 2005a). It has been hypothesized that hCG injection in the vulvar submucosa would enhance the action of this hormone by acting more rapidly on the ovaries, delaying the absorption or slowing the metabolism of this hormone and extending its action, while allowing a reduction in the minimal effective dose (Meira et al. 2006). Since the application of hCG via IVSM is much more difficult to apply than the IM injection, our results point out to the IM route as a better option from a practical standpoint to widespread its use under field conditions. The overall number of follicles of different sizes prior to experimental treatments (−15, −10 and −5) were pooled and are presented in Table 2 as '−5 days'; the observed small, medium and large follicles across treatments are presented for days 1 and 5, also in Table 2. The number of small, medium and large ovarian follicles was not different (P > .05) among goats at −15, −10 and −5 days (5.8 ± 1.0, 1.4 ± 0.2 and 2.0 ± 0.5 mm, respectively) and was indicative of a reduced ovarian activity without follicles capable of achieving ovulatory status. These follicles have been described by Ginther and Kot (1994) as the dynamic pool of antral follicles that develop and regress rather than be part of a cohort of gonadotrophindependent follicles, one or more of which might grow to the pre-ovulatory size and ovulate. Then, the remaining follicles undergo structural and functional atresia at various stages of development, under the influence of gonadotrophins (Gonzalez-Bulnes et al. 2005;Simoes et al. 2006). Nonetheless, after hCG administration (days 1-5), the number of small follicles was decreased (IM: 2.9 ± 0.7 and IVSM: 3.3 ± 0.6 for day 1 and IM: 2.8 ± 0.4 and IVSM: 2.3 ± 0.4 for day 5), while no differences occurred with respect to the number of medium (IM: 2.4 ± 0.7 and IVSM: 1.5 ± 0.3 day 1) and large-sized follicles (IM: 2.3 ± 0.3 and IVSM: 2.6 ± 0.7 for day 5) between the hCG groups. Interestingly, however, by day 5, while the control group depicted an  increased number of small follicles, those goats treated with hCG depicted a reduction in the number of small follicles as well as a concomitant increase in the number of large follicles, irrespective of the hCG administration route (Table 1). Therefore, goats receiving the hCG treatment depicted the greater population of larger follicles, suggesting an increased follicular steroidogenesis which apparently augmented the plasma oestradiol concentration, and promoted an increased oestrus behaviour in the hCG-treated goats, similar to that reported by Fonseca et al. (2005b). Apparently, the rise in gonadotropin induced the gonadotrophin-dependent follicles to emerge from a pool of gonadotrophin-responsive follicles (Viñoles et al. 1999). After emergence, one or several of these follicles are able to achieve an ovulatory status by expressing lutenizing hormone receptors on their granulosa cells and become independent of FSH influence. The fact that exogenous hCG increased the number of large follicles indicates that hCG is a feasible mechanism to stimulate folliculogenesis in anoestrus goats, and that these potentially ovulatory follicle(s) continued to develop and ovulate. Ovulation occurred when follicles reached nearly 8 mm in diameter (Table 1). No differences (P > .05) in follicle diameter were observed between groups treated with hCG (IM: 7.6 ± 0.2 and IVSM: 7.8 ± 0.2). The mean diameter of the dominant follicle was similar to that reported in previous studies carried out under similar conditions (Lehloenya et al. 2008;Vazquez et al. 2010), and was indicative of fertility as confirmed by the observed pregnancy rate (IM: 66% and IVSM: 55%) and litter size for both IM and IVSM groups (1.5 ± 0.2 and 1.6 ± 0.2, respectively) ( Table 1).
To conclude, our results indicate that a single injection of 100 IU hCG may be a simple and effective way of inducing fertile oestrus and suitable reproductive outcomes, the last irrespectively of the administration route, either IM or IVSM. These simple methods generated important results regarding not only oestrus induction and ovulation, but also interesting out-ofseason values for pregnancy rate, kidding rate and litter size, particularly since the control group was unable to depict oestrus and become pregnant. It is important to highlight that such reproductive outcomes were obtained in a genotype mainly based in quite seasonal mix-breed dairy breeds (Native × Alpine-Saanen dairy) under marginal conditions.

Disclosure statement
No potential conflict of interest was reported by the authors.