Introduction

An increase in the ovulation rate and prolificacy in small ruminants has been achieved in an efficient way through hormone treatments and by increasing the energy content in the diet (flushing). Although the flushing mechanism is not yet fully understood, the evidence indicates that its effects are produced at the ovarian level; furthermore, they are independent of gonadotropin concentrations and are associated with increases in the blood glucose and insulin levels (^{Downing et al., 1995}; ^{Muñoz-Gutiérrez et al., 2004}; ^{Dupont et al., 2014}).

In sheep, short-term supplementation with lupin grain, a legume with a high metabolizable energy and protein content, can increase ovulation rates. Short-term supplementation with lupin grain stimulates folliculogenesis and increases ovulation rates, without inducing changes in body weight and condition (^{Scaramuzzi et al., 2006}). Glucose and insulin may influence the ovulation rate by stimulating follicle development, resulting in a higher number of follicles capable of responding to gonadotropin stimulus (^{Gutiérrez et al., 1997}; ^{Viñoles et al., 2005}). Furthermore, glucose and glucosamine stimulate follicle steroidogenic capacity by increasing insulin-like growth factor type I (IGF-I) activity (^{Muñoz-Gutiérrez et al., 2004}). Studies performed in goats show that an increase in insulin, either induced by diet or by insulin administration, increases the ovulation rate (^{Suguna et al., 2009}; ^{Zabuli et al., 2010}).

The administration of glycogenic solutions can cause an increase in the blood glucose and insulin concentrations. In sheep, short-term treatments with glycerol and propylene glycol have been used to increase ovulation rates. ^{Rodriguez Iglesias et al. (1996)} were able to increase the ovulation rate in seasonally anovulatory ewes through the oral administration of 100 mL of a glycerol and propylene glycol solution, which were administered immediately before ram exposure. ^{Gutierrez et al. (2011}) observed increased serum glucose concentrations, insulin, and ovulation rates with a single oral dose of 300 mL of glycerol during the induction of luteolysis with PGF2α in Pelibuey sheep. In the same study, a similar effect on the ovulation rate with a single oral dose of 100 mL of glycerol was achieved.

Moreover, insulin promotes early embryonic development. In vitro insulin exerts a mitogenic and an antiapoptotic effect on embryos (^{Byrne et al., 2002}; ^{Augustin et al., 2003}), resulting in an increased proportion of embryos reaching the blastocyst stage. Additionally, high insulin concentrations negatively affect embryonic development in cattle (^{Fouladi-Nashta et al., 2006}) and sheep (^{Carrera-Chávez et al., 2014}). Furthermore, a positive effect from insulin on luteal function has been observed. In a goat study, insulin was associated with higher concentrations of progesterone during pregnancy (^{Suguna et al., 2009}). High levels of progesterone are associated with accelerated embryonic development (^{Garret et al., 1988}), which could translate into fewer embryonic losses. In dairy cows, the administration of 1 L of oral glycerol during the first 6 days post-insemination increases blood insulin levels and increases the pregnancy rate (^{Ortega et al., 2010}). Therefore, this study tested whether the oral administration of 100 mL of glycerol, at the time of progestin removal and in the first 6 days of the estrous cycle, increased the ovulation rate, prolificacy, and pregnancy rate in goats.

Materials and methods

Treatments

In all goats, an intravaginal sponge impregnated with 45 mg of fluorogestone acetate (FGA; Chronogest; MSD Animal Health, Mexico; Intervet México, S.A. de C.V., Huixquilucan, Estado de México, México) was inserted and remained in situ for 12 days. When the sponge was removed, a luteolytic dose of sodium cloprostenol (Celosil; MSD Animal Health, Mexico) was administered. Upon sponge withdrawal, the goats were randomly assigned to one of the following treatment groups: the glycerol (n = 65) group, which received an oral drench of 100 mL of glycerol repeated on days 0, 2, 4, and 6 following estrus (estrus = day 0), and the control (n = 64) group, which did not receive glycerol. Males fitted with an apron performed estrus detection. Females in estrus were mated to males of proven fertility.

Between days 8 and 12 post-mating, the ovulation rate was determined via a transrectal ultrasound exam by counting the number of corpora lutea; for this purpose, a 7.5 MHz linear transducer (Aloka, Co., Ltd., Tokyo, Japan) was used, which was adapted to a rigid support to allow for handling (^{Gutierrez et al., 2011}). The pregnancy rate was determined by ultrasonographic pregnancy diagnosis on day 40 after service. Prolificacy was determined at birth (Figure. 1).

Figure 1: A schematic overview of the treatments. 

Results and Discussion

The oral administration of 100 mL of glycerol at the time of progesterone withdrawal did not increase the ovulation rate, and the proportion of goats with multiple ovulations or multiple births was similar between treatments (Tables 1 and 2). The ovulation rate [glycerol= 1.84 ± 0.64 vs control=1.80 ±0.72 (mean ± standard deviation)] and the prolificacy [glycerol= 1.58 ± 0.61 vs control=1.60 ±0.64 (mean ± standard deviation)] were similar between treatments. These results are different from those observed in sheep by ^{Rodriguez Iglesias et al. (1996)} and ^{Gutierrez et al. (2011)}. The reason the treatment did not have a favorable effect on the ovulation rate may be related to the metabolic response to the oral glycerol doses. The 100 mL glycerol dose was determined based on the results obtained in sheep by ^{Gutierrez et al. (2011)}; in their study, the administration of 100 mL glycerol caused an increase in the ovulation rate similar to the one obtained with 300 mL. For this reason, the administration of 100 mL was chosen. In the present study, serum insulin concentrations were not affected by the treatment (P = 0.11), but there was an interaction between the treatment and time (P = 0.08). Thus, insulin concentrations tended to be higher within 2 and 4 h post-treatment (Figure 2). Likely, this slight increase in insulin concentrations was not enough to improve the ovulation rate. The cause of the variation in response to the same glycerol dose observed between sheep and goats is unknown. Studies evaluating glycogenic solutions have been performed in sheep and cattle, but not in goats (^{Rodriguez-Iglesias et al., 1996}; ^{Ortega et al., 2010}; ^{Gutierrez et al., 2011}). Although these 3 species are ruminants, they present differences in their digestive function and their use of forages. Goats have physiological adaptations that allow them to leverage high fiber feeds more efficiently (^{Silanikove, 2000}). Therefore, they may be less sensitive to variations in the quality and quantity of offered feeds. Oral glycerol is used as a substrate in rumen fermentation from which propionate can be obtained. However, it can also be absorbed directly as glycerol through the rumen mucosa, which will be converted later into glucose in the liver (^{Trabue et al., 2007}). Although this metabolic pathway is the same in ruminants, its efficiency in triggering insulin secretion may be different between species; this could explain, in part, the success of the treatment in increasing the ovulation rate in sheep as well as its failure in goats. The results of this study promote the development of future studies in goats to evaluate different glycerol doses, until the dose that triggers an optimal response in serum insulin is determined.

Table 1: Percentage of goats with multiple ovulations that received an oral drench of 100 mL glycerol. 

Table 2: Percentage of goats kidding more than one kid that received an oral drench of 100 mL glycerol. 

Figure 2: Average insulin concentrations (± standard error) in goats treated with 100 mL of glycerol () and controls (). Treatment by time interaction: P = 0.08. 

Furthermore, this study proposed that the oral administration of glycerol during the first days of embryo development (days 0, 2, 4, and 6 of the estrous cycle) favors embryonic survival, which would increase the proportion of pregnant goats and prolificacy. This hypothesis was based on the expected effect of glycerol on insulin concentrations and the effects of insulin on embryonic development. Studies performed in vitro and in vivo show that insulin improves embryonic development by increasing cell proliferation and decreasing apoptosis (^{Augustin et al., 2003}; ^{Suguna et al., 2009}). Meanwhile, low insulin concentrations adversely affect embryonic development and interferon tau production (^{Thatcher et al., 1995}). Furthermore, in dairy cows, an increase in insulin concentrations induced by the oral administration of 1 L of glycerol during the first 6 days of embryonic development (days 0, 2, 4, and 6) has been shown to improve pregnancy rates (^{Ortega et al., 2010}). In the present study, however, the pregnancy rate (glycerol = 88 vs control = 85%) and the proportion of multiple births were similar between treatments (P > 0.05; Table 2). The lack of effect on prolificacy and pregnancy rate observed in this study was probably due to the marginal increase in insulin concentrations after the oral administration of 100 mL of glycerol. Furthermore, the pregnancy rate obtained in both groups was high, which makes it more difficult to identify a positive effect from any treatment aimed at improving fertility. Parity was observed to have an effect on ovulation rate and prolificacy; however, there was no interaction between the treatment and parity (Tables 1 and 2; P > 0.1).

The administration of oral glycerol during the first days of the estrous cycle may favor embryonic development and conception rate by increasing serum progesterone concentrations. This hypothesis is based on studies in cattle in which insulin increases circulating progesterone concentrations (^{Spicer y Echternkamp, 1995}; ^{Cooke et al., 2012}). In this study, nonetheless, progesterone concentrations between 1 and 17 days post-mating were similar between treatments, and no interaction was observed between the treatment and time (P > 0.05; Figure 3).

Figure 3: Average concentrations of progesterone (± standard error) in goats treated with 100 mL of oral glycerol () and controls () (P > 0.05). 

The good body condition of the goats used in this study, as well as the season in which the treatment was applied, may have influenced the lack of a positive response to glycerol administration. The goats received a diet that met the NRC requirements, and they had a body condition between 2 and 3 at the beginning of the experiment, which is reflective of the conditions associated with a high pregnancy rate and high prolificacy and are similar to those obtained consistently within the flock. Conversely, this study was completed during the height of the reproductive period, which contributed to the excellent response in fertility. Future experiments should be conducted in conditions in which the fertility and prolificacy are diminished, such as in cyclicity induction programs during anestrus or in marginally nourished goats; in these circumstances, the supplementation proposed in this study may improve fertility. Treatments exist that do not increase pregnancy rates globally, but rather do so in subgroups of animals. In a previous study conducted by our group, for example, bovine recombinant somatotropin only increased pregnancy rate in goats in anestrus, as well as only increased prolificacy in primiparous goats, who naturally tend to have a lower pregnancy rate and prolificacy (^{Hernández y Gutiérrez, 2012}).

Conclusions

In conclusion, the oral administration of 100 mL of glycerol at the time of progestin withdrawal and in the first six days of the estrous cycle did not increase the ovulation rate, prolificacy, or pregnancy rate in goats.