Increasing the litter size is of great importance to goat keepers and thus in goat breeding prolificacy is one of the economically important traits. In reproduction, KISS1 and the NGF genes play a key role. KISS1 gene is responsible for multiple type of birth and could be considered as a candidate for selection of reproductive traits
(Messager et al., 2005; Richard et al., 2008; Gaytan et al., 2009; An et al., 2013; Clarke et al., 2015). KISS1 has also shown to regulate seasonal reproductive activity in Syrian hamsters and sheep
(Revel et al., 2006; Revel et al., 2007; Clarke et al., 2009). Significant increase (P<0.05) of KISS1 mRNAs was associated with puberty goats
(Samir et al., 2018). Similarly, NGF gene and its relationship with reproductive traits such as superior ovulation rate and litter size in farm animals have been studied to aid in the selection of multiparous animals
(Menchaca et al., 2002; Akpa et al., 2011, Naicy et al., 2016). NGF plays a critical role in development of the mammalian ovary, oogenesis and follicular synthesis
(Lavasani et al., 2006; Julio-Pieper et al., 2009). Previous researcher has associated higher expression levels of ovarian NGF mRNA with prolificacy in goats
(Naicy et al., 2016). The interaction between NGF and gonadotrophins in bovine oviduct was reported where NGF could increase the expression of FSHR and LHR in oviduct epithelial cells
via its effects on its receptor
i.
e. NTRK1
(Li et al., 2014). Although the importance of KISS1 and NGF for reproduction is apparent, these genes have not been properly evaluated as a candidate for genetic reproduction in animals, especially in prolific small ruminants. Therefore, the current study was aimed to determine the relative expression of mRNA expression of KISS1 and NGF genes in the ovaries of two Indian goat breeds with differing litter size.
The present investigation was conducted in two different goat breeds i.e. Barbari and Jamunapari maintained at the ICAR-Central Institute for Research on Goats (CIRG) Makhdoom. Barbari goat (1.51) is relatively more prolific than the Jamunapari goat (1.34)
(Chauhan, 2016). The female goats with age group of 2-5 years were selected for sample collection. Ovary tissues were collected, washed twice in phosphate buffer saline and stored in RNAlater (Sigma) at -70°C for future use. The total RNA was isolated using Trizol method and quality was checked by UV VIS photometer (Eppendorf). The first strand cDNA was synthesized using Transcriptor first strand cDNA synthesis kit (TAKARA). Real Time PCR analysis was carried out in Light Cycler 480 (Roche Applied Science, Indianapolis, IL, USA) using SYBR Green® master mix (TAKARA) as per manufacturer instructions. A set of primers for KISS1 gene (F 5’-TGAACGTGCTGCTTTCCT-3’ and R 5’ GCCTGTG GTTCTAGGATTCTC-3’) and for NGF gene (F-5’ GGGAGA GGTGAACATCAACAA-3’andR-5’-GTCGTGGTACAATAC GAGTTCC-3’) were used. Two endogenous reference genes namely GAPDH (F-5’-GCAAGTTCCACGGCACAGTC’-3 and R-5’ -CCC ACTTGATGTTGGCAGGA-3’)andβ-actin(F-5’ TGCCCTGAGGCTCTCTTCCA-3’ and R 5’ -TGCGGATG TCGACGTCACA-3’) were used to normalize the gene expression of KISS1 and NGF genes. The thermal profile was standardised as initial denaturation at 94°C for 10 min, followed by 45 cycles, denaturation at 94°C for 10 sec, annealing at 62°C for 15 sec, and extension at 72°C for 20 sec. All the samples were taken in duplicate and a non-template control was run simultaneously. Fold-change in expression level of the target genes were presented using E-method
(Livak and Schmittgen, 2001). The crossing point (Cp) readings for each unknown sample were then used to calculate the amount of either the target or housekeeping gene using the second derivative maximum method with the Light cycler 480 analysis software version 1.5 (Roche Applied Science, Indianapolis, IL, USA). The geometric mean of the resulting ratios was calculated,
e.g. T1/R(all)=(T1/R1×T1/R2)1/2 and T2/R(all) = ( T2/R1×T2/R2)½
Where,
T- Target gene,
R- Reference gene
The relative ovarian tissue abundance of KISS1 and NGF mRNA among two breeds were determined using quantitative real time PCR. The relative expression of KISS1 and NGF genes was higher in ovaries of Barbari than Jamunapari goats (Fig 1).
The expression pattern of the KISS1 gene revealed that Barbari and Jamunapari had 2.275 and 0.007 fold gene expressions, respectively, than the control. Similarly, the expression pattern of the NGF gene indicated that the Barbari breed had higher expression (3.237 fold) than the control while the Jamunapari breed had 1.749 fold expression .The cross point (CP) value showed substantial effect (P<0.05) among the breeds. The expression levels of KISS1 and NGF genes between the Barbari and Jamunapari goats were markedly different. Many researchers have shown the association of KISS1 gene polymorphism with litter traits in goats
(An et al., 2013; Sahoo et al., 2019). Various earlier studies identified that Kisspeptin regulate GnRH and gonadotropin secretion and the gene expression of KISS1 has important role in animal reproduction
(Navarro et al., 2005; Silveira et al., 2010; Navarro et al., 2012; Choe et al., 2013; Vargas et al., 2017). Our finding of higher ovarian expression of NGF gene was in agreement with earlier report where NGF expression in ovaries of goats with history of multiple births had higher than singles in goats
(Naincy et al., 2016). The finding of the current investigation implies the importance of KISS1 and NGF genes in goat reproduction and consequently prolificacy.
