Effect of Organic Zinc Supplementation on Semen Production and Quality in Kankrej Bulls

Zinc is an essential micro-mineral for animal life with the concentration of 20-50 mg/kg body weight in livestock. It has a wide range of biological functions. Zinc is involved in cell replication and differentiation, particularly in nucleic acid metabolism throughout the life. It is also responsible for production, storage and secretion of hormones, maintenance of immune system and electrolyte balance (Mcdonald et al., 2007). It is found in high concentration in the male reproductive tract as well as in semen (Chia et al., 2000) and plays a very important role in male fertility as it acts as hormone receptor modulator for testosterone in Leydig cell leads to maintenance of sperm production and quality (Roy et al., 2013; Kaliky et al., 2019). Zn deficiency affects the DNA synthesis in the germ cell, sperm formation and also reduce the sperm motility (Yamaguchi et al., 2009). Forty eight percent of Indian soils are deficient in Zn (Arunachalam et al., 2013) which may affect the male reproduction. Thus, Zinc deficiency is one of the major factors for infertility in breeding bulls. Kankrej is proud native breed of Banaskantha region of Gujarat. Semen producing ability and quality of individual bull is highly essentials to ensure the supply of superior quality germplasm for maintaining the production as well as the conservation of indigenous breeds. Recent studies also have supported the fact that supplementation of zinc has improved qualitative as well as the quantitative aspects of semen quality in bulls (Thakur et al., 2019; Majumder et al., 2020). So, a study was undertaken to study the effect of organic zinc supplementation on semen parameters in Kankrej bulls.

The use of the animals and the experimental procedure in present trial was approved by the Institutional Animal Ethics Committee (IAEC) constituted as per the Article 13 of the rules of the Committee for the Purpose of Control and Supervision on Experiments on Animals (CPCSEA), laid down by the Government of India. The IAEC approval number was VETCOLL/IAEC/2019/14/PROTOCOL-06.

Location of study, experimental animals and feeding
 
An experiment was carried out at Livestock Research Station of Sardarkrushinagar Dantiwada Agricultural University, Sardarkrushinagar, Banaskantha, Gujarat (Latitude 24.35°N, longitude 72.59°E and Mean Sea level l89). Eight Kankrej bulls (Age 4-6 years) under semen collection were selected as experimental animals. A total of 128 ejaculates (64 pre-supplementation and 64 post-supplementation) were collected from the bulls. They were fed on basal diet during pre-supplementation period, while Zinc Propionate @ 100 ppm/animal was supplemented to basal diet as a source of zinc (KemTRACE Zn; Kemin Industries) during post-supplementation for a period of 60 days. Dose of 100 ppm was selected by taking into consideration weight of bulls and previous records of experiments. The animals were fed as per ICAR (2013) feeding standards to meet the nutrient requirements. The data of dry matter intake of the bulls during experimental period is given in Table 1. All the bulls were kept in a well-ventilated shed having a cemented floor and arrangements for individual feeding. Clean and fresh drinking water was provided twice daily i.e., at 9.00 am and 2.00 pm to all the animals.
 
Proximate composition of feeds and fodder
 
Representative sample of feeds and fodder were analyzed (AOAC, 1999) for proximate composition. Zinc (ppm) for different feedstuffs was calculated using standard reference values.
 
Collection of semen and evaluation of its quantitative and qualitative characteristics
 
The semen from all the bulls was collected in an artificial vagina over a dummy or male partner for initial pre-supplementation period of two months followed by two months of experimental zinc feeding. Then, 8 ejaculates from each animal were collected to study the effect of Zn supplementation on semen characteristics. Two months feeding was carried out intentionally to complete one cycle of spermatogenesis (for sperm formation and maturation) which is 60 days in bulls. All semen samples were evaluated for quantitative and qualitative characteristics.
 
Evaluation of semen quantitative characteristics
 
Ejaculate volume (mL) of semen was recorded to the nearest 0.1 mL in a graduated glass tube. The concentration of sperm (millions/ml) in the fresh semen was determined using a photometer (Accucel, IMV, France) against 530 nM wave length.
 
Evaluation of semen qualitative characteristics
 
Mass motility of semen was graded from a 0-5 scale, based on the appearance of waves and swirls created by sperm movement when visualized by keeping one drop of semen on a glass slide, without cover slip, under low power microscopic magnification (10x) (Salisbury et al., 1978). Extremely rapid waves or swirl motion of sperms were given a scale of 5, comparatively slower waves and swirls were given a 4 numerical scale and likewise further slow moving, extremely slow moving, no movement and non-motile sperm were given 3, 2, 1 and 0 numerical scales, respectively. The individual motility of freshly diluted semen was assessed after covering a semen drop on a glass slide with a thin cover slip at 37°C, under high power magnification (40x). Live sperm percentage was calculated by using Eosin-Nigrosin stain. The same stain was utilized for counting abnormal sperm percentage.
 
Statistical analysis
 
The data were analyzed for statistical significance using “paired- t test” (Snedecor and Cochran, 1994).

Proximate composition of feeds and fodders
 
The proximate composition of feeds and fodders fed to the experimental animals is given in Table 1. Nutrient and of concentrate (BIS Type II), oat forage and jowar hay was within the range as reported by (Joshi et al., 2021). Zinc (ppm) content of different feeds was also calculated and was found to be within normal range (Chaudhary et al., 2017). Dry matter intake Average dry matter intake/100 kg body weight of experimental animals pre and post supplementation was 2.46 and 2.54.


 
Semen quantitative attributes
 
The average semen quantitative and qualitative attributes of Kankrej bulls are presented in Table 2. Overall mean values for semen volume were 3.99±0.19 and 3.81±0.16 ml per ejaculate in pre- and post-supplementation, respectively, the difference was non-significant (P>0.05) among groups. However, mean values for sperm concentration were significantly (P<0.05) higher in post supplementation group (2162.36±112.0 million/ml vs 1325.68±80.08) as compared to pre-supplementation groups. Zinc has an indispensable role in male spermatogenesis. The production of any cell including sperm necessitates extensive mitotic or meiotic cell division which requires DNA polymerase and RNA polymerase. Both of these are Zn dependent enzymes for their activity. Zn also helps in encoding a transcription factor involved in spermatogenesis and stimulates production of testosterone from seminiferous tubules. Zinc containing metalloenzymes like sorbitol dehydrogenase and lactate dehydrogenase are also very important enzymes involved in the process of spermatogenesis (Kumar et al., 2006). All these factors may account for improved sperm concentration and sperm number per ejaculate in Zn-supplemented group.

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Semen qualitative attributes
 
The average semen qualitative attributes of Kankrej bulls are presented in Table 3. The average mass motility of sperms was significantly (P<0.01) higher during post-supplementation (3.92±0.02%) when compared to the pre-supplementation (3.78±0.03%). Mean initial motility of sperms was 76.08±0.70 and 78.33±0.41% in pre- and post-supplementation, respectively, the difference being significant (P<0.05). The overall live sperms were significantly (P<0.01) higher during post-supplementation (73.33±0.91%) when compared to the pre-supplementation (63.53±1.03%). The average abnormal sperms were significantly (P<0.01) reduced during post-supplementation (5.35±0.45%) than the pre-supplementation (6.83±0.58%). Mean post-thaw motility of sperms were significantly (P<0.01) higher during post-supplementation (51.14±0.86%) as compared to the pre-supplementation (46.17±1.40%).

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Flagella help in the sperm motility which in turns depends on energy supply in the form of ATP. Zinc helps in energy utilization of sperm through stimulation of ATP cleavage by prostasomes in semen (Ronquist et al., 2013). Zn is also a scavenger of free oxygen radical and protects sperm from oxidative damage and lipid peroxidation by inhibiting phospholipase (Kumar et al., 2006). Kumar et al., (2006) found increased live spermatozoa (%) in Zn supplemented group in crossbred bulls. Sabhapati et al., (2015) also observed increased live spermatozoa (%) in Zn supplemented group in Karan Fries during supplemented period. 

It may be concluded that additional supplementation of 100 ppm of zinc propionate in the diet of Kankrej bulls improved semen quality in terms of quantitative and qualitative characteristics.

The authors express thankfulness to the Research Scientist, Livestock Research Station, SDAU, Sardarkrushinagar and the Principal, College of Veterinary Sciences and Animal Husbandry, KU, Sardarkrushinagar for providing necessary facilities to carry out the experiment.

This work was supported by the College of Veterinary Sciences and Animal Husbandry, KU, Sardarkrushinagar.

There is no conflict of interest.