Efficiency of Copper and Selenium during Estrus Synchronization on Estrus Behaviour, Estradiol and Progesterone Concentration in Salem Black Goats

The goat is considered as “poor man’s cow” and provides livelihood for billions of low and medium input farmers (Gupta et al., 2019). As India is an agricultural based country, goats are predominantly distributed in India with a population of 148.88 million which constitutes to 27.80% of the total livestock population (Basic Animal Husbandry Statistics, 2019). The increased demand for goat production is attributed to their regular supply of milk, meat and cashmere. Although, estrus synchronization in goats with progesterone (Pietroski et al., 2013, Hashemi and Safdarian, 2018), estradiol benzoate (Souza-fabjan​ et al., 2017), double PGF2α injections and Ovsynch protocols (Riaz et al., 2012) have been adopted to improve goat population, the estrus response was not always 100% and the conception rates were below 70% (Menchaca et al., 2007; Riaz et al., 2012). There is further necessary to tighten the existing synchronization protocols with novel substances that could modulate ovarian function. This research gap is of a greater challenge to the scientific community in the increasing population scenario to enhance the livestock production.

Trace minerals such as copper (Garcia-diaz et al., 2012; Roychoudhury et al., 2014; Soni et al., 2018) and selenium (Basini and Tamanani et al., 2000; Gabryszuk et al., 2002; Koyuncu and Yerlikaya 2007; Yao et al., 2018) were largely found to regulate ovarian granulosa cell estradiol synthesis and estrus behaviour in ruminants. Copper supplementation in vivo significantly increased estradiol, progesterone, FSH and LH concentration in Simmental heifers (Wang et al., 2008). Similarly, selenium supplementation in vivo significantly increased follicular growth (Vazquez-hernandez​ et al., 2017), estrus response (Gabryszuk et al., 2002; Koyuncu and Yerlikaya 2007; Wani et al., 2016) and conception rate (Ganie et al., 2014; Musa et al., 2018) in ruminants. The existing evidences are based on prolonged dietary supplementation and/or multiple parenteral administrations of copper and selenium, while studies are meager on single parenteral administration of copper and selenium in combination with PGF2α during estrus synchronization protocols. In view of these considerations an attempt has been made to evaluate the effect of parenteral administration of copper and selenium in comparision to GnRH on estrus behaviour and changes in estradiol and progesterone hormone concentration in Salem black goats.

Animal management
 
The present study was conducted in the year 2017-2018 in an experimental livestock unit, ICAR-National Institue of Animal Nutrition and Physiology (NIANP), Adugodi, Bengaluru, Karnataka, India. Thirty adult female Salem black goats of twelve to eighteen months of age were selected. The animals were housed in a well-ventilated shed and maintained under proper hygienic conditions. The animals were fed as per ICAR feeding standards (Ranjhan, 1998).
 
Experimental design
 
Prior to the start of the experiment, all the animals were injected with cloprostenol 250 μg/animal (Pregova, Virbac) to lyse the corpus luteum present if any. Ten days (day 0 of the experiment) after the first injection, the second injection of cloprostenol was injected and the animals were alloted to the following groups. Group I (GI, n=6) served as the control, Group II (GII, n=6) animals were given intramuscular injection of 4 µg of Buserelin, GnRH analog (Pregulate, Virbac). Group III (GIII, n=6) animals were given intramuscular injection of cupric chloride (Himedia) @ 20 mg per animal. Animals in Group IV (GIV, n=6) were given intramuscular injection of sodium selenite (Sigma Aldrich) @ 10 mg per animal. Groups V (GV, n=6) animals were given intramuscular injections of cupric chloride @ 20 mg plus sodium selenite @ 10 mg per animal. The experiment was replicated thrice with a gap of 60 days and the same grouping was followed for each experiment.
 
Estrus detection
 
Estrus was determined initially by signs such as frequent tail wagging, bleating, swollen vulva and vulval mucous discharge. Further confirmation was made by exposing the does to apronised bucks. The female does which stand still on being mounted were considered to be in estrus. Estrus percent was calculated by total number of animals responded in a group divided by total animals of animals in a group. Since the experiment was replicated thrice the percent estrus response of three experiments were used for statistical analysis. The onset and duration of estrus was also recorded.
 
Blood collection and estimation of plasma estradiol and progesterone concentration
 
Whole blood was collected from day 0 to day 7 from all the animals. The plasma was separated by centrifugation at 3500 rpm for 15 min at room temperature and stored at -20°C for estimation of estradiol and progesterone hormone concentration. Plasma estradiol (Tsang et al., 1980) and progesterone (Radwanska, 1978) concentration was determined by enzyme linked immunosorbent assay (ELISA) using a microplate reader (Thermo Scientific) by ELISA kits (Cal biotech kits).

Statistical analysis 
 
The data obtained on various parameters were statistically analyzed using Graphpad PRISM software 6.0. The differences between means of different groups were analyzed by one way ANOVA and multiple comparisons were performed by Tukey’stest. The percentage values of estrus response were converted to arcsine values before statistical analysis and significance was determined at P<0.05.

Trace minerals hold critical roles in maximizing reproductive efficiencies in ruminants. The trace elements likely to be of most practical significance are copper, selenium and zinc (Pal, 2015). It has been established that copper has a stimulatory effect on the release of GnRH and FSH (Rouchoudhary et al., 2016) which triggers the synthesis and release of estradiol. Furthermore, copper inpart may promote ovarian granulosa cell estradiol synthesis via mRNA expression of Cytochrome P450 Family 19 subfamily A member 1(CYP19A1) which encodes the enzyme aromatase that converts androgens to estradiol in the ovarian granulosa cells (Soni et al., 2018; Tej et al., 2021). Selenium was found to stimulate ovarian granulosa cell estradiol synthesis via increase in the number of follicles (Vazquez-hernandez​ et al., 2017) granulosa cell proliferation (Basini and Tamanini, 2000), increased activity of antioxidant enzymes, reduce lipid peroxidation (Said et al., 2012) and upregulation of genes involved in estradiol synthesis signalling pathways (Yao et al., 2018). These evidences suggest that, trace minerals such as copper and selenium could be possibly used to improve estrus response in synchronization protocols.
 
Effect of copper and selenium on estradiol concentration
 
Within-group between days comparison (Fig 1) revealed no significant (p>0.05) increase in estradiol concentration from day 0 to day 7 in all the groups. Between group comparison (Fig 2) revealed significantly higher (p<0.05) estradiol concentration in GnRH group compared to other groups, while the estradiol concentration did not differ significantly (p>0.05) between control, copper, selenium, copper plus selenium groups. The significant (p<0.05) increase in estradiol concentration in GnRH group compared to other groups could be attributed to GnRH injection which stimulates FSH secretion from the anterior pituitary and subsequent follicular growth and estradiol synthesis from granulosa cells (Marques et al., 2018). No significant (p>0.05) effect of copper and selenium on estradiol concentration was observed in the present study. Contradictory to the present findings, dietary supplementation of copper sulphate (Wang et al., 2008) significantly increased estradiol concentration in ruminants. Moreover, oral supplementation of sodium selenite in ewe (Sen et al., 2011) and selenium rich yeast in Taihang black goats (Shi et al., 2018) significantly increased estradiol concentration. The significant increase in estradiol concentration with copper and selenium in other studies could be due to prolonged dietary supplementation. The estradiol concentration observed in the present study was in agreement with other reports where a similar estradiol concentration of 7.7±1.7 pg/ml in Dwarf goats (Khanum et al., 2008) and 5.19±0.71 pg/ml in Huanghuai Goats (Pang et al., 2009) was observed.



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Effect of copper and selenium on progesterone concentration
 
Within group between days comparison (Fig 3) revealed that the progesterone concentration was significantly higher (p<0.05) on day 0 (before PGF₂α injection) in all the groups and the concentration decreased significantly (p<0.05) on day 1 and continued to maintain at significantly (p<0.05) lower levels till day 5 in all the groups. Between groups comparison (Fig 4) revealed significantly (p<0.05) higher progesterone concentration in GnRH group compared to other groups, while the progesterone concentration did not differ significantly (p>0.05) between control, copper, selenium, copper plus selenium groups. A significant (p<0.05) decline in progesterone concentration from day 0 to day 1 could be due to PGF₂α injection which selectively lyses the developing corpus luteum (CL) (Omontese et al., 2016) and subsequent decrease in progesterone concentration. A significant (p<0.05) increase in progesterone concentration in the GnRH group compared to other groups could be attributed to GnRH injection which would have stimulated LH secretion and subsequent progesterone synthesis from luteal cells (Marques et al., 2018). The study revealed no significant (p>0.05) effect of copper and selenium on progesterone concentration in Salem black goats. Contradictory to our findings dietary supplementation of copper sulphate (Wang et al., 2008) significantly increased progesterone concentration in ruminants. Moreover, oral supplementation of sodium selenite (Kamada et al., 2014), selenium (Cerny et al., 2016) selenium enriched yeast (Shi et al., 2018) significantly increased progesterone concentration in ruminants which may be due to prolonged dietary supplementation of copper and selenium. The progesterone concentration observed in the present study was in agreement with other reports where a progesterone concentration of 0.1 to 5.4 ng/ml in Dwarf goats (Khanum et al., 2006), 0.1±0.03 to 7.7±0.6 ng/ml in Dwarf goats (Khanum et al., 2008) and 0.51±0.10 to 10.72±0.71 ng/ml in Markhoz Goat (Farshad et al., 2008) was observed respectively.

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Effect of copper and selenium on estrus response, onset and duration of estrus
 
The mean±SEM values of onset, duration and estrus response were presented in Table 1. There was no significant (p>0.05) difference in onset, duration and estrus response between groups.  In the estrus cycle of the goat, at the end of diestrus phase PGF₂α secreted from the uterine endoemetrium lyse the CL, lowering the progesterone concentration. This reduced progesterone triggers the secretion of GnRH from hypothalamus and subsequent secretion of FSH from anterior pituitary. Under the influence of tonic FSH secretions the existing dominant follicle of the follicular wave continuous to secrete estradiol which induce estrus behaviour and when the estradiol concentration reaches peack it triggers ovulation (Fatet et al., 2011). In the current study, low estrus response was noticed in control groups (PGF₂α alone). Similar to our findings, a low estrus response of 4.3-15.8% in Red Sokoto goats (Tauheed et al., 2010), 30% in Persian downy does (Hashemi and Safdarian, 2018) and 0% in Salem black goats (Gupta et al., 2019) was observed in goats synchronized with double PGF₂α protocol. Relatively, a high estrus response of 50% was observed in GnRH group. Similar estrus response was observed in our previous study on Salem black goats where GnRH based Ovisynch Protocol was used (Gupta et al., 2019). This effect may be attributed to GnRH injection which would have directly trigerred hypothalamic-pituitary-ovarian axis. Furthermore, low estrus response was observed in copper and selenium groups. No similar studies were conducted so far to compare our results but selenium when injected with sponge impregnated with progesterone it resulted in 90% (Qureshi et al., 2010), 88% (Wani et al., 2016) and 100% (Mujawar et al., 2019) estrus response in buffaloes.



The onset of estrus observed in the present study fall in line with other studies where double PGF2α injection resulted in onset of estrus of 45-45.5 h in Black bengal does (Khandoker et al., 2009) and 47.7±10.1 h in Alpine, Saanen and Toggenburg cross (Esteves et al., 2013). The finding of the present study was in agreement with other reports where goats synchronized with PGF₂α showed a duration of estrus of 25-51h in Black bengal does (Khandoker et al., 2009) and 26.8±15.0 h in Alpine, Saanen and Toggenburg cross (Esteves et al., 2013) respectively. The onset and duration of estrus observed in the present study falls within the range as that of other breeds of goats and this information is important for the determination of the time of mating and or insemination to maximize reproductive efficiency in goats.

In the present study, copper and selenium did not significantly affect estradiol, progesterone, estrus response, onset and duration of estrus, while GnRH injection significantly improved estrus associated responses compared to other groups. Whereas, from other studies it was evident that selenium and progesterone combination resulted in high estrus response in buffaloes (Qureshi et al., 2010; Wani et al., 2016; Mujawar et al., 2019). The findings are suggestive of use of copper and selenium in combination with GnRH/progesterone devices to evaluate the effect on estrus associated responses.  Moreover single injection of minerals just before estrus might have not improved estradiol synthesis significantly which may be due to the limitations imposed by other competing factor (Landau, 2000) as copper and selenium are involved in diverse functions, the given dose would have utilized for general body functions. While the significant effect of copper and selenium in other studies could be due to prolonged dietary supplementation and or multiple injections.

Although copper and selenium were proved to be estrogenic in ruminants, in the current study no significant effect was noticed. It was concluded that single parenteral injection of copper and selenium along with PGF₂α did not improve estrus behavior, estradiol and progesterone concentration in Salem black goats. Whereas GnRH was proved to be effective in improving the reproductive efficiency in goats. Therefore, the current study strongly hints a necessity for the use of combintion of GnRH/progesterone along with trace minerals. However, in the present study no such combinations were used. Further studies are focussed on use of copper and selenium in combination with GnRH/progesterone devices.

The authors are greatly thankful to the Director, National Institute of Animal Nutrition and Pysiology (NIANP), for provision of facilities at the laboratories and livestock farm.

The authors declare that they have no competing interests.