​Evaluation of Level of Pesticide Residue in Seminal Fluid of Crossbred Bulls

Pesticides act as endocrine disruptors and cause huge disturbances in steroidogenesis, spermatogenesis and exhibition of sexual behavior. Due to inadvertent use of potentially dangerous pesticides, dairy animals are highly vulnerable to their exposure via soil, food, water and animal-derived oil or fat products incorporated into processed feeds (Ratnakaran et al., 2014). These pesticides include not only old and biologically persistent organochlorine pesticides like dichloro diphenyl trichloroethane (DDT), hexa chlorocyclo hexane (HCH), heptachlor, aldrin and endosulfan, but also highly toxic organophosphorus pesticides (OPP) like phosphamidon, chlorpyrifos (CPF) and malathion. These toxicants modulate and/or disrupt reproductive and hormonal milieu by acting at a variety of sites including hypothalamus, pituitary and reproductive organs (Zama and Uzumcu 2010). Only few studies have been conducted to assess the pesticide levels in blood of dairy cattle/buffaloes, however no information is available related to breeding bulls. In view of the above information, the present study was designed to evaluate the level of pesticide residues in seminal plasma of crossbred breeding bulls to assess the impact of pesticide levels on semen quality of crossbred breeding bulls.

Experimental design
 
Nineteen fresh semen ejaculates were collected from Milkfed, Khanna and PLDB (Punjab Livestock Development Board), Nabha, Punjab and stored at -80^oC. Immediately after collection, the semen was centrifuged at 4500 × g for 20 min at 4^oC. The supernatant (seminal plasma) was stored at -20^oC until assay. Pesticide residues were evaluated using gas chromatography (GC) and Gas chromatography-mass spectrometry (GC-MS) (Kamarianos et al., 2003a) (Table 1) in the Pesticide Residue Testing Laboratory, School of Veterinary Public Health, Guru Angad Dev Veterinary and Animal Sciences University, Ludhiana, Punjab in 2019-2020.
 

 
1. Estimation of pesticide residues in semen samples
 
Sample treatment
 
Each sample was extracted with methanol and centrifuged after thawing at temperature and once homogenised. The extract was next passed through a solid phase extraction cartridge filled with 500 mg of C18 guaranteed porous silicon oxide, Analytichem Bond Elut LRC from Varian (Harbour city, CA, USA), to separate and concentrate the analytes, per the theme shown in Fig 1 (Guardino et al., 1996) with slight modifications. To carry out this step, the extracts were classified in batches of 10. Each and every batch was treated by suggests that of a vacuum process station (Analytichem vacation Elut SPS24, Varian) at a consistent flow-rate of 2 ml/min. The extracts were processed simultaneously, but the flow was controlled individually and, for each sample, the process was stopped by means of a valve when the extract came level with the surface of the solid phase inside the cartridge. The elutes were collected in pre-weighed vials so as to control the final volume, after concentration using nitrogen flow. Each sample batch was analyzed along with a blank of methanol following constant process.
 

 
Estimation using gas chromatography
 
Gas chromatography detection was used for the pesticide residues in all the analyzed commodities. Gas chromatography is a technique used for separating and analyzing compounds that can be vaporized without decomposition. The compound must have sufficient volatility and thermal stability. In gas chromatography, the mobile phase is a carrier gas, usually an inert gas such as helium or a non-reactive gas such as nitrogen. The stationary phase is a microscopic layer of liquid or polymer on an inert solid support, inside a piece of glass or metal tubing called a column. Fully programmed auto injector injects the sample using micro syringe through a rubber septum into a flash vaporizer port at the head of the column. Once in the column, the compounds in the mixture are separated by differences in the positions of adsorption, equilibrium between the gaseous components of the sample and the stationary phase. In present study, cleaned up extracts were analyzed by GC equipped with ECD for OCPs, SPs and FTD for OPs compounds with standardized operating conditions (Table 2).
 

 
Sample introduction to GC
 
The cleaned up extract measuring 1-2 µl was injected in GC similarly as standard solution through auto injector. Residues were quantitatively determined by comparison of the retention time (Table 3) and peak areas/heights of the sample chromatogram with those of standard solutions run under the same operating conditions. The concentration of pesticide residues was reported as ng g^-1 on fresh weight basis. The formula for quantification of residues:
 

  2. Post thaw semen quality (Post-thaw motility, viability and membrane integrity) in relation to pesticide residues of crossbred breeding bulls
 
Methodology
 
Nineteen fresh semen ejaculates, from Milkfed, Khanna and PLDB (Punjab Livestock Development Board), Nabha, Punjab were collected and stored at -80^oC. The post thaw semen quality was assessed in Department of Veterinary Gynaecology and Obstetrics, Guru Angad Dev Veterinary and Animal Sciences University, Ludhiana, Punjab.
           
Individual sperm motility and sperm viability were estimated in fresh ejaculates and after thawing using standard procedure and semen was subjected to Hypo-Osmotic Swelling Test (HOST) for plasma membrane integrity (PMI) and for HOST following procedure was used:
a.   Two hypo-osmotic solutions were prepared as follows: 2.7% aqueous solution of fructose (1.351 gm/50 ml distilled water) and 1.47% aqueous solution of sodium citrate (0.735 gm/50 ml distilled water).
b.   Equal volumes of both solutions (0.5 ml each) were mixed and kept in an incubator at 37oC for 10 minutes.
c.   50 μl of semen was added in above hypo-osmotic solution (Fructose + Sodium citrate) and incubated at 37^oC for 30 minutes.
d.   10μl of this mixture was taken on glass slide and covered with glass cover.
e.  Slide was observed under 10 X objective lens to determine the number of spermatozoa showing swollen head and coiled tail indicating sperms with intact plasma membrane (HOS positive sperm).
f.    Total hundred spermatozoa were counted to determine the percentage of HOST positive spermatozoa.

Pesticide residue in semen of crossbred breeding bulls
 
In the present study on crossbred breeding bulls, the samples were analyzed for the pesticide residues. Seven OCP were screened namely heptachlor epoxide, chlordane, fipronil, lindane, methoxychlor, op-DDT, endrin. Eleven OPP were screened namely chorpyrifos, dichlorovos, ethion, monocrotophos, malathion, parathion-methyl, profenphos, phorate, triazophos, quinalphos and phosalone and four SP’s namely cypermethrin, permethrin, deltamethrin, cyalothrin were screened in the semen samples and none of the semen samples were found positive for any of the above pesticide residues. This may be attributed to the feeding of fodder cultivated at the stations itself where pesticides were not used, therefore no pesticide residue found in the semen samples of the crossbred breeding bulls. Also, the use of biologically persistent OCPs (DDT and HCH) on crops is banned worldwide due to their potential for bioaccumulation, trans-boundary movement and biological effects (Thullner 1997). In this study, OPP used were not detectable in the semen samples of animals. This could be attributed to the blood-testis and epididymis barrier (Kamarianos et al., 2003a) and short biological half-life of OPPs (Smith 1995). The graphs for the sample analysis are presented (Fig 2-5).
 

 

 

 

 
Evaluation of semen
 
Semen evaluation of crossbred bulls is presented in Table 4. In present study, individual sperm motility (was found to be significantly higher (p<0.05) in fresh semen (90.21±4.31%) than the post thaw (57.89±5.23) semen. Similar observation was also reported by Kumar et al., (2008).
 

 
Individual motility (%)
 
In present study, individual sperm motility was found to be significantly higher (p<0.05) in fresh semen (90.21±4.31%) than the post thaw (57.89±5.23) semen. Similar observation was also reported by Kumar et al., (2008).
 
The results of the present study for initial motility are higher than those reported by Angasaria et al., (2002). They observed initial motility in the range of 30-83% with an average of 67.24±2.59 and 66.80±2.28, in HF crossbred bulls. In a previous study, the mean initial motility was recorded to be 67.60±0.47 (%) in HF crossbred bulls (Kanchan and Matharoo 2015). The results in the present study for initial motility is higher than Vyas et al., (1992) who reported 60.69±1.43 (%) initial motility in HF crossbred bulls. Shaha et al., (2008) found 56.6 to 76% initial motility in HF crossbred bulls. Hossain et al., (2012) observed 63.7% initial motility in 97 breeding bulls at the Central Cattle Breeding and Dairy farm, Savar, Dhaka. Another study reported 78.2, 75, 80 and 80 (%) initial motility in Pure HF, 50% cross, 75% cross and Borena bulls, respectively at NAIC, Kaliti, Addis Ababa, Ethiopia (Alemu et al., 2014). In another study, the initial motility was found to be 60.39±0 % in HF crossbred bulls (Chauhan et al., 2017). The variation in the initial motility may be due to variation between individual bulls, percent live sperms, method of detection (CASA and manual), season and technician.
       
The results of post thaw motility (%) in present study (57.89±5.23) are slightly higher than those reported by Mathur et al., (2014)  who found the mean post-thaw motility in exotic breeds (HF and Jersey) was 52.40% followed by 50.85% percent in crossbreds (HF, Jersey and other crosses). The overall mean post-thaw motility was 51.02%. They reported that, among the semen production stations, the average post- thaw motility of frozen semen varied from 52.70 to 49.70%. In another study, it was reported that 53.8% of tested bulls exhibited >40% post thaw motility in 14 cross-bred and 12 pure bred bulls (Zodinsanga et al., 2015). Gopinathan et al., (2016) observed 49.8±0.0% post thaw motility in crossbred Holstein Friesian bulls. The variation in the post thaw motility might be due to variation between individual bulls, cryopreservation techniques, period of storage, thawing methods and maintenance of liquid nitrogen.
 
Viability (%) and total sperm abnormalities (%)
 
In present study, viability (%) of sperms were found to be significantly higher (p<0.05) in fresh (75.57±5.31) as compared to post thaw (70.17±4.64) semen of bulls. Also, the abnormality (%) of sperms was found to be significantly lower (p<0.05) in fresh (13.61±3.71) than post thaw (18.22±3.59) semen bulls. The results of the present study are in slight concurrence with previous study (Vyas et al., 1992). They reported that the abnormal spermatozoa rate was 9.58±0.86 initially in fresh semen sample and 14.07±0.87 after thawing in frozen semen sample in HF crossbred bulls. Soren et al., (2016) observed 10.74±0.18% major sperm abnormalities in adult healthy Karan Fries bulls and found that they were increased in hot-humid as compared to spring at Animal Breeding Research Centre (ABRC) of National Dairy Research Institute (NDRI), Karnal. The variation in the abnormal spermatozoa rate may be due to season, affection of testis and defects during preparation of slides.
 
Membrane integrity (%)
 
In present study the HOST (%) was found 67.23±4.76 in fresh semen which decreased to 46.82±3.55 in post thaw semen.The results of the present study for hypo osmotic swelling test are slightly higher than Zubair et al., (2013). They reported that the mean sperm positive to HOST was 27% in crossbred semen, 40% in Frisian semen and 47% in Sahiwal semen. Zodinsanga et al., (2015) reported that 53.8% of tested bulls exhibited >35% hypo-osmotic swelling response analyzed from frozen-thawed semen of 14 cross-bred and 12 pure bred bulls. The results of present study for HOST are lower than Soren et al., (2016). They reported the value for Hypo-osmotic swelling test to be 59.75±0.57 and 64.97±0.84% in the summer and spring seasons, respectively, in Karan Fries bulls from Animal Breeding Research Centre (ABRC) of National Dairy Research Institute (NDRI), Karnal. Chauhan et al., (2017) reported HOS test value of 73.76±0.47% Frieswal bulls. The variation in the HOST may be due to variation in percent osmolarity, method of HOST, cryopreservation damages to spermatozoa.