Association of SSCP Polymorphisms of HSP70 Gene with Physiological, Production and Reproduction Performance in Sahiwal and Crossbred Cows

Ever increasing human population is exerting tremendous pressure on natural resources including livestock (Herrero and Thornton, 2013). Changing global environment is also posing a major threat to the sustainability of livestock production systems (Naqvi and Sejian, 2011; Polsky and Keyserlingk, 2017 and Rojas-Downing et al., 2017). Global warming impairs production and reproduction performance, metabolic health and immune competence of livestock.
       
Selection and breeding methods are the only tools available for animal breeders to meet these challenges. Though crossbreeding was proved to be the fastest way of increasing milk production, adaptability of crossbreds was poor as reflected by lower breeding efficiency. Further, crossbreds are found to be highly susceptible to diseases and less tolerant to heat stress (Singh, 2016). Current strategies focussing on managemental manipulations to mitigate the effects of heat stress are partially successful and are only short term measures (Berman, 2005). Feed and housing modifications with focus on genetic manipulations could be a sustainable long term strategy to reduce the effects of heat stress (Boonkum et al., 2011; Scholtz et al., 2013).
       
Heat shock proteins (HSPs) are molecular chaperones that play a critical role in recovering cells from stress and cytoprotection, protecting the cells from subsequent insults. Through their ability to recognize nascent polypeptides, unstructured protein areas and exposed hydrophobic stretches of amino acids, they protect stressed cells. Chaperones thus hold, translocate or refold denatured proteins and prevent their irreversible aggregation with other cell proteins (Archana et al., 2017).
       
Genes encoding the heat shock proteins are called HSP genes and their nomenclature is given by the HUGO Gene Nomenclature Committee. Though there are many HSP genes, thermo tolerance is mainly correlated with HSP70 and HSP90 genes in Livestock species. HSP70 is reported to be the most abundant and temperature-sensitive playing a crucial role in environmental stress and thermal adaptation (Gade et al., 2010). In farm animals elevated levels of expression of proteins of the HSP70 and HSP90 family were observed in sheep, buffalo, cattle, broilers and goats during the summer season (Archana et al., 2017). Polymorphism in the HSP70 and HSP90 genes have shown an association with heat tolerance, milk production, fertility and disease susceptibility in livestock (Shergojry et al., 2014; Kumar et al., 2015, Bhat et al., 2016). They can make ideal candidate gene markers for the selection of animals with better climate resilience, immune response and superior performance. (Hassan et al., 2019).
       
Although differences in thermo tolerance at physiological and cellular level are documented (Collier et al., 2006; Chaiyabutr et al., 2008; Wilson and Crandall, 2010 and Dalcin et al., 2016) in both Bos indicus and Bos taurus cattle, information on polymorphism of HSP genes in Sahiwal cattle and Holstein Friesian crossbreds is scarce. There are few reports from India regarding the association of HSP70 and HSP90 gene polymorphism with heat tolerance in Tharparkar cattle (Bhat et al., 2016), Deoni cattle (Kerekoppa et al., 2015), Jersey crossbred cows (Sailo et al., 2015) and from abroad in Holstein cow (Li et al., 2011). Therefore in the present study, polymorphisms of HSP70 and their correlation with various physiological, production and reproduction traits in Sahiwal and crossbred cows were explored.

Experimental animals
 
A total of 50 Sahiwal cows maintained at the Livestock Farm Complex, College of Veterinary Science Rajendranagar and 50 crossbred cows (Holstein Friesian × Sahiwal crosses with 7/8 exotic inheritance maintained at the Military Dairy Farm, Secunderabad were utilized for the present investigation.
 
Weather conditions
 
Hyderabad, the capital city of Telangana State is located at 17.366°N Latitude and 78.476°E Longitude. It is situated at an elevation of 536 meters (1607 feet) above the mean sea level. The data on the weather conditions, maximum and minimum temperatures (°C), dry and wet bulb readings (°C) and relative humidity (%) during the experimental period were collected from Agriculture Climate Research Center, ARI, Hyderabad. During the present study year of 2018, the average environmental temperatures ranged from 27.92°C in December which was the coldest month to 41.24°C in May, which was the hottest month.  Hyderabad experienced moderate rainfall in July, August and September. The average annual rainfall received was 766 mm and the average relative humidity was around 65% varying from 45% during summer to 78% during the monsoon. Winters were moderately cold with temperatures ranging from 15°C to 31°C.
 
Genomic DNA isolation
 
About 10 ml of peripheral blood was collected aseptically from each of the representative cows from the external jugular vein into a sterile vacutainer tube containing 0.5 percent EDTA. Genomic DNA of each of the animals was isolated from the blood samples using the standard phenol-chloroform extraction method as described by Green and Sambrook (2012), with minor modifications. The purity of genomic DNA samples was assessed by measuring the optical densities (OD) at 260 nm and 280 nm against blank using Nanodrop (Thermo Fisher Scientific). The samples which gave the OD ratio between 1.7 and 1.9 were assessed as good and used for PCR amplification.
 
Physiological parameters
 
Data about the physiological parameters, respiration rate (RR) and rectal temperature (RT) of each animal under the present study were recorded twice daily for 30 days in each of the three seasons i.e during May (2018) for summer, August (2018) for rainy and from mid-December (2018) to mid-January (2019) for winter season respectively and the average was taken as final reading for each cow in association analysis. The timings of recording the physiological parameters were 8 AM and 2 PM. The Heat Tolerance Coefficient (HTC) based on respiration rate and rectal temperature was calculated for each animal using the formula given by Benezra (1954).
 
Production and reproduction traits
 
Data on each animal about different aspects like animal no., sire no., dam no., date of birth, date of calving, lactation length and lactation milk yield, etc., were collected from the history sheet/daily farm registers maintained at the concerned farms. The various production and reproduction traits like total lactation milk yield (TLMY), peak yield (PY), lactation length (LL), service period (SP), dry period (DP) and calving interval (CI) were calculated from the available data in both Sahiwal and crossbred cows.
 
PCR primers and amplifications
 
Two pairs of primers (procured from BioServe Biotechnologies Pvt Ltd, Hyderabad) specific for the desired regions (295 and 220 bp) of the initial coding region of HSP70 gene as available in the literature (Bhat et al., 2016) were used to amplify the targeted regions. The details of primer sequences, length of the primer (bp), melting temperature (Tm) are presented below.


       
The PCR reactions were carried out on a total of 12.5 µl volume containing template DNA of 1 µl (50-100 ng/µl), 1.0 µl  each of forward and reverse primers (10pM), 2.5 µl of 10X Taq buffer, 0.8 µl of dNTPs (10 mM), 0.125 µl of Taq Polymerase (5 units/µl) and 6.075 µl  of Nuclease free water. Amplification was done in a pre-programmed thermo cycler (Prima-Duo, Himedia labs). The PCR cycling conditions involved an initial denaturation at 95°C for 5 min, followed by 35 cycles with initial denaturation at 95°C for 30 sec, annealing temperature of 50°C for 35 sec, extension at 72°C for 30 sec followed by a final extension at 72°C for 10 min. PCR products were detected by electrophoresis on 2% agarose gel stained with ethidium bromide.
 
Single strand conformation polymorphism (SSCP)
 
The presence of variation in the fragments of HSP70 gene was screened using the single-strand conformation polymorphism (SSCP) technique using the amplified PCR products. Polymorphism was identified basing on the band pattern observed in the SSCP gels after silver staining (Bassam et al., 2007). The different patterns observed were recorded for further analysis. The most common band pattern identified was named as A. If there are more bands, in addition to the common bands, they were marked as B, C, etc., depending on the band pattern.
 
Genotype and allele frequencies
 
Genotype frequencies for variant genotypes were calculated using the formula:
   
Allele frequencies were calculated as follows:
 
Where,
AA and BB = Genotype frequencies of homozygotes.
AB = Genotype frequency of heterozygote.
A and B = Allele frequencies.
 
Association analysis
 
Statistical analysis was performed to study the association of each SSCP genotype on physiological, production and reproduction traits in Sahiwal and Crossbred cows. Data on physiological traits was corrected for season effect and used for association analysis. The univariate GLM model of SPSS 25 was used to perform the analysis according to the following statistical model:
 
Where,
Y_ijk   = Dependent variable (respiration rate, rectal temperature, heat tolerance coefficient, total lactation milk yield, peak yield, lactation length, gestation period, service period, dry period and calving interval).
µ  =  Overall mean.
G_i  =  Effect of i^th SSCP genotype (i= 1…n).
P_j   =  Effect of j^th parity of the animal at the time of blood collection (j= 1…n).
e_ijk=   Random error assumed to be distributed normally and independently with mean zero and variance σ²_e.

Significant differences between the means of different genotypes and parities were tested by Duncan’s multiple range test (DMRT). Values were considered significant at P£0.05 and presented as means±standard errors.

The representative image showing the PCR amplified products of Fragment I and Fragment II of HSP70 gene, showing the sizes of 295 bp and 220 bp are presented in Fig 1 and 2 respectively, while the PCR-SSCP polyacrylamide gel images are presented in Fig 3 and 4 respectively. The Fragment I (295 bp) of the HSP70 gene was found to be polymorphic in both Sahiwal and crossbred cows. In Sahiwal, two SSCP genotypes namely AA and AB were documented whose frequencies were estimated to be 0.58 and 0.42, respectively while in crossbred cows, two SSCP patterns AA and AC were documented with frequencies 0.62 and 0.38, respectively (Fig 3).
 

 

 

       
The Fragment II (220 bp) fragment of HSP70 gene was found to be monomorphic in both the breeds studied  (Fig 4) and hence, was not considered for further association analysis.
 

       
The means obtained from the statistical analysis for the effect of HSP70 fragment I genotypes on the physiological, production and reproduction traits in Sahiwal and crossbred cows are presented in Table 1, 2 and 3 respectively.
 

 

 

 
Association analysis of HSP70 gene polymorphism with physiological traits
 
The SSCP genotypes of Fragment I of HSP70 gene had no significant effect on physiological parameters studied in both Sahiwal and crossbred cows. However, Bhat et al., (2016) found allele A of HSP70 to have positive correlation with thermal tolerance and genotype AA demonstrated superior heat tolerance. Various other authors also suggested that polymorphism in the HSP70 gene could be used in genetic improvement programmes of cattle for heat tolerance (Cai et al., 2005; Li et al., 2011; Deb et al., 2013 and Sodhi et al., 2013).
 
Association with production traits
 
The SSCP genotype AA of Fragment I of HSP70 gene had significantly higher peak yield of 10.77 kg in Sahiwal cows and higher total lactation milk yield (3005.01 kg) in crossbred cows. The present investigation established a relationship between the HSP70 Fragment I genotypes and production traits, with allele A having a positive effect on milk yield.  It may be suggested that AA genotype may be incorporated in marker-assisted selection to increase milk yield in both Sahiwal and crossbred cows. Rosenkrans et al., (2010) also reported polymorphisms in the promoter region of the HSP70 gene, some of which were associated with the milk yield in crossbred Brahman cows which may be useful in selecting cows with greater fertility.
 
Association analysis with reproduction traits
 
The SSCP genotypes of Fragment I of HSP70 gene had a non-significant effect on the reproductive traits studied in both Sahiwal and crossbred cows except on service period in crossbreds. Crossbred cows with AA genotype had a longer service period. Rosenkrans et al., (2010) also reported polymorphisms in the promoter region of the HSP70 gene, some of which were associated with calving rates in crossbred Brahman cows which may be useful in selecting cows with greater fertility.

The present study revealed that the different SSCP genotypes of the 295 bp fragment of HSP70 gene were associated with production and reproduction traits and indicated their possible role in marker-assisted selection (MAS). However, the SSCP patterns obtained in the present study and their association analysis with physiological parameters did not reveal significant differences among the genotypes indicating that a larger population of cows with a wide genetic base may be needed to elucidate the association of polymorphism.