Effect of a single-reduced dose of equine follicular fluid on the timing of oestrus and reproductive performances in goats

Abstract

To evaluate the use of equine follicular fluid (EFF) in a single-reduced dose on the synchronisation of oestrus in Boer×Nubian goats, the following experimental treatments were applied: Goats in group T1 (FGA, n=10) were synchronised in oestrus by intravaginal sponges impregnated with 45 mg FGA (Chronogest-MSD) for 12 days; group T2 (T2, FGA + eCG, n=16) goats were synchronised as in T1, plus 400 IU eCG, 48 h before sponge withdrawal; T3 goats (FGA + PGF₂α, n=11) received the sponge as T1 plus an i.m. dose of PGF₂α (1 ml, Prosolvin-MSD) 48 h before sponge withdrawal; T4 (FGA + EFF, n=19) animals received the sponge as T1 plus 2 ml EFF at sponge withdrawal and finally, group T5 (FGA + PGF₂α + EFF, n=10) was like T3 plus 2 ml EFF at sponge withdrawal. Presentation: onset and duration of oestrus were recorded, and fertility, prolificacy and fecundity were calculated. All females showed oestrus. The onset of oestrus was different (P < 0.01), T4 and T5 goats showing a delay (50.5±4.9 and 48.2±2.0 h for T4 and T5 groups, respectively). Litter size (2.10±0.11 kids/kidding) and fecundity (1.90±0.23 kids/goat) were higher (P<0.01) in T5 goats compared to T1 and T4, with no differences with T2 and T3. The combination of EFF with FGA and PGF₂α in goats produces a delayed onset of oestrus and improves prolificacy and fertility in comparison with the use of FGA and EFF.

Keywords:

• goat
• equine follicular fluid
• oestrus
• synchronisation
• reproduction

1. Introduction

Goat reproduction and, in particular, those strategies focused on the control of the sexual cycle, have been the aim of several research groups in the last decades (Argüello 2011). It includes both non-hormonal, (male effect; Rincón et al. 2011), and hormonal techniques (progestagens, prostaglandins, melatonin; Abecia et al. 2011). A new alternative is the use of follicular fluid (FF), which is steroid-free and rich in inhibin secretion (de Jong and Sharpe 1976), which has been deemed as a glycoprotein hormone with endocrine and paracrine actions to regulate FSH secretion from the pituitary and follicular development from the ovaries of some mammals (Medan et al. 2007). It has been used unprocessed, as a source of inhibin (Knight et al. 1991), producing negative feedback on FSH secretion, which in turn affects follicular development and, as a consequence, litter size (Knight and Glister 2001). A negative relationship between FSH secretion and follicular inhibin in goats has been described (Medan et al. 2003; Ganguly et al. 2010). Exogenous administration of follicular fluid causes an inhibition of follicular development at the initial phase and after inhibin metabolisation, the pituitary releases FSH, resulting in an increment of its growth and ovulation rate (Wallace and McNeilly 1985). Moreover, FSH hypersecretion after the end of a treatment with follicular fluid has resulted in an increase of follicular development (Miller and Martin 1993). Both bovine (Beard et al. 1989) and buffalo (Palta et al. 1996) follicular fluids are rich in inhibin, which produce a delay in the onset of oestrus induced by prostaglandins and increase ovulation rate in goats during the breeding season (Ghosh et al. 2005) and seasonal anoestrus (Das et al. 2005). Hernández et al. (1997) observed that equine follicular fluid (EFF), steroid-free, inhibits FSH secretion during seasonal anoestrus in sheep and delays a synchronised oestrus with PGF₂α during the breeding season, playing the same role as inhibin. Regarding the use of FF as a treatment to control the sexual cycle, it has been applied under different protocols from different species: buffalo FF in goats (Das et al. 2009); bovine FF in sheep (Miller and Martin 1993), and EFF in sheep (Hernández et al. 1997).

EFF is easy to obtain in some countries like Mexico, where there are no problems collecting it at the slaughterhouse. Because of its ability to delay the preovulatory LH peak and inhibit FSH secretion, it could be a cheap alternative to synchronise oestrus in goats in developing countries, where hormonal treatments are less extended at commercial level due to their prices. Thus, this experiment has been designed to test whether or not EFF can be used as a means to control the sexual cycle in goats, with or without an increment of reproductive parameters. To reach this objective, a treatment based on the use of EFF has been associated and compared with traditional hormonal methods such as fluorogestone acetate (FGA) and prostaglandin F2α (PGF₂α) under tropical conditions.

2. Materials and methods

The experiment was carried out at the goat experimental flock of the Postgraduate College at Montecillo, Mexico state (19°29′N, 98°54′W), Mexico, during the breeding season (September–October). In order to determine the ovarian cycle, one month before the onset of the experimental procedures, blood samples were collected weekly to determine plasma progesterone concentrations by RIA (DPC). Sensibility of the assay was 50 pg mL⁻¹, with an intra and inter variation coefficient <12%. Plasma progesterone levels >1 ng mL⁻¹ indicated ovarian activity.

Sixty-six adult Boer×Nubia goats (3.0±0.6 years old), with a mean live weight of 48.00±2.72 kg, were randomly allocated into five experimental treatments (T1–T5). Goats in group T1 (FGA, n=10) were synchronised in oestrus by intravaginal sponges impregnated with 45 mg FGA (MSD) for 12 days; group T2 (T2, FGA + eCG, n=16) goats were synchronised as in T1, plus 400 IU eCG, 48 h before sponge withdrawal; T3 goats (FGA + PGF₂α, n=11) received the sponge as T1 plus an i.m. dose of PGF₂α (1 ml, Prosolvin-MSD), 48 h before sponge withdrawal; T4 (FGA + EFF, n=19) animals received the sponge as T1 plus 2 ml EFF at sponge withdrawal and finally, T5 groups (FGA + PGF₂α + EFF, n=10) was like T3 plus 2 ml EFF at sponge withdrawal.

EFF was obtained from mare ovarian follicles after slaughter from a slaughterhouse. Thus, ovaries (6–8 cm long and 3–4 cm wide) were collected and transported to the laboratory; follicles were measured with a calliper and those with a diameter of at least 20 mm (mean 27±6.2 mm) were identified, and the follicular fluid was aspirated by puncture with an 18G needle. A positive correlation between follicular inhibin concentration and oestradiol levels with follicular size has been observed (Campbell et al. 1991). After EFF extraction, it was homogenised and distributed in 10 ml tubes, to be centrifuged at 3200 rpm for 15 min. The supernatant was sterilised by filtration and an antibiotic was added as indicated by Hernández et al. (1997). Finally, it was frozen at −20°C until use.

Onset of oestrus was determined by a harnessed buck to avoid fertilisation, which was introduced with the goats 24 h after sponge withdrawal, every 2 h for 30 min. Oestrous detection was finished when the last goat presented oestrus. Bucks were allocated in pens adjacent to goats, so that visual, olfactive and auditive contacts between genders were allowed through the experiment. A goat was considered in oestrus when it accepted mounting and oestrous duration was deemed as the interval until the end of mounting behaviour. Goats in oestrus were mounted by the bucks 12 and 24 h after oestrous onset. Fertility rate (percentage of pregnant goats in each group), prolificacy (number of kids born/ kidding) and fecundity (number of kids born/goat) were calculated.

Onset and duration of oestrus, prolificacy and fecundity were compared by ANOVA. When the model was statistically significant, a multiple comparison of means (±SE) was performed by LSD. The percentage of goats presenting oestrus and fertility were compared by χ² or Fisher exact tests, as appropriate.

3. Results

Plasma progesterone concentrations revealed that all the goats included in the trial presented reproductive activity at the onset of the experiment.

Oestrus was shown by 100% of the goats in the experiment. Oestrous onset was delayed in goats treated with EFF (T4 and T5) in comparison with T1, T2 and T3 (P<0.05) (Table 1), although mean oestrous length was 35.6±3.9 h, with no differences between groups (Table 1). Fertility rate was similar between groups, although both prolificacy and fecundity were significantly affected by treatments (Table 2). Thus, prolificacy was lower in T1 goats compared with the rest of the treatments, with T5 animals presenting differences with T4 (P<0.05). Fecundity was higher in T5 when compared with T1 and T4.

Table 1. Onset and oestrous length (mean±S.E) in goats treated with a sponge impregnated with fluorogestone acetate (FGA) for 12 days (T1), combined with equine chorionic gonadotropin (eCG) 48 h before sponge withdrawal (T2), prostaglandin F2α (PGF2α) 48 h before sponge withdrawal (T3), equine follicular fluid (EFF) at sponge withdrawal (T4) or PGF2α 48 h before sponge withdrawal and EFF at sponge withdrawal (T5).

Treatment	n	Oestrous onset (h)	Interval (h)	Oestrous length (h)	Interval
T1, FGA	10	38.7±4.4^a	21.4–68.7	35.7±4.0	21.4–48.0
T2, FGA + eCG	16	37.3±5.5^a	23.3–69.5	35.8±5.5	28.0–45.0
T3, FGA + PGF2α	11	33.5±1.0^a	30.0–39.0	35.3±2.0	24.0–41.0
T4, FGA + EFF	19	50.5±5.0^b	29.0–59.6	36.0±4.6	21.0–47.0
T5, FGA + PGF2α +EFF	10	48.2±2.0^b	32.0–44.0	35.4±4.3	4.0–45.0
Total	66	41.64 ± 3.6	27.1–56.2	35.6±3.9	19.7–45.2
Note: Different superscripts in the same column indicate significant differences between treatments (P<0.01).

Table 2. Fertility (percentage of pregnant ewes), prolificacy (number of kids/goat kidding) and fecundity (number of kids/goat) in goats treated with a sponge impregnated with fluorogestone acetate (FGA) for 12 days (T1), combined with equine chorionic gonadotropin (eCG) 48 h before sponge withdrawal (T2), prostaglandin F2α (PGF2α) 48 h before sponge withdrawal (T3), equine follicular fluid (EFF) at sponge withdrawal (T4) or PGF2α 48 h before sponge withdrawal and EFF at sponge withdrawal (T5).

Treatment	n	Fertility (%)	Prolificacy	Fecundity
T1, FGA	10	60	1.30±0.21^a	0.80±0.25^a
T2, FGA + eCG	16	75	2.08±0.08^b	1.56±0.24^b
T3, FGA + PGF2α	11	91	2.00±0.00^b	1.82±0.18^b
T4, FGA + EFF	19	74	1.78±0.11^bc	1.32±0.20^ab
T5, FGA + PGF2α + EFF	10	90	2.10±0.11^bcd	1.90±0.23^bc
Total	66	77.3	1.90±0.06	1.47±0.11
Note: Different superscripts in the same column indicate significant differences between treatments (P<0.01).

4. Discussion

Under the conditions of this experiment, the combination of EFF with eCG and/or PGF2α to synchronise oestrus in goats produced a delay in its onset and increased prolificacy and fecundity. This treatment could be an alternative to the complicated management previously described in sheep and goats. Thus, Das et al. (2009) used three steroid-free buffalo FF ml, in goats at 12 h intervals for 3 days; Miller and Martin (1993) used two bovine FF ml in sheep, every 8 h the last four days of the luteal phase or Hernández et al. (1997), who injected sheep with i.v. 3 ml EFF every 8 h for 5 days in the seasonal anoestrus and every 3 days during the breeding season.

The delayed oestrous onset observed in this experiment has been previously observed in sheep also receiving EFF (Hernández et al. 1997) and in goats treated with buffalo FF (Das et al. 2009). However, no evidence of this fact had been reported after a single-reduced EFF dose in goats. The content of inhibin described in bovine FF (Tortonese and Gomez-Brunet 1996; Turzillo and Nett 1997) and in EFF (Hernández et al. 1997) exerts negative feedback on FSH secretion, limiting follicular growth, and likely to be responsible for the delay of oestrous onset. Das et al. (2009) reported that the caprine ovary, after being treated with buffalo FF, responded by increasing the number of antral follicles, which is associated with external signs of oestrus (Das et al. 2005). It is thought that the single-reduced dose of EFF used in this experiment had high inhibin content as the goats that did not receive any EFF did not show any delay (T1, T2 and T3). However, the use of PGF2α induced an earlier presentation of oestrus, which was also observable in T4 and T5 goats, probably due to the presence of a corpus luteum in their ovaries. In fact, goats treated with PGF2α showed less variation in the occurrence of oestrus.

Oestrous length was similar between groups, which mean that the inclusion of EFF in the synchronisation treatment produces a similar degree of synchronisation than treatments based in FGA and eCG or FGA and PGF₂α, in agreement with Regueiro et al. (1999).

Although T1 ewes presented fertility rates 14–30% lower than the rest of the groups, no statistical differences were observed, perhaps due to a low number of goats per treatment. Prolificacy was significantly affected by treatment (P<0.01), goats in the T5 treatments presenting the highest number of kids born per kidding. Similarly, this group produced the highest number of kids per goat (fecundity), since their fertility rate was 90%. Thus, the inclusion of EFF to control oestrous cycle in goats is not a limiting factor for ovulation, and even when combined with FGA and PGF2α, this could be an interesting way to increase kid production. Considering that inhibin exerts negative feedback on the anterior pituitary to limit FSH secretion (Hernández et al. 1997; González-Bulnes et al. 2004), the metabolisation of the inhibin content of the administered EFF could propitiate increased follicular development (Das et al. 2009), which in turn could increase ovulation rate (Glencross 1992), such as when goats are immunised against inhibin (Dietrich et al. 1995). In treatment T5, it is likely that PGF2α lyses corpora lutea (CL) of the ovary, so that the progesterone blocking on LH secretion disappears, and medium-size follicles restart their growth.

Undoubtedly, the actions of other hormones such as oestradiol, activin or follistatin, could have affected the response. Since the role of inhibin is exerted through its secretion at ovarian level, followed by its transport through blood circulation, the action of the other hormones seem to depend on paracrine interactions at the pituitary level. Therefore, follistatin reduces FSH secretion together with activin, blocking its action (Evans 1999).

In conclusion, EFF in combination with FGA and PGF2α was able to increase kid production in comparison with other hormonal treatments. In order to develop a more accurate protocol of oestrous synchronisation including EFF, it is necessary to establish adequate procedures of collecting follicular fluid from the slaughterhouse to standardising the age of mares, follicle size, season, etc. Moreover, further studies involving the follicle dynamic should be carried out to fully understand the effect of EFF in combination with progestagens and prostaglandins.

Acknowledgements

To the ‘Mejoramiento al Profesorado’ programme (PROMEP, SEP) and the ‘Facultad de Medicina Veterinaria y Zootecnia’ of the ‘Benemérita Universidad Autónoma de Puebla’, México, and the ‘Colegio de Postgraduados’.