Cross Breeding Promotes Deterioration of Semen Quality in Cattle Bulls

Ibrar Muhammad Khan1, Rifat Ullah Khan1,*, Muhammad Subhan Qureshi2, Tahir Usman2, Adnan Khan1, Zakir Ullah1 and Hamid Rehman1 1Department of Animal Health, Faculty of Animal Husbandry and Veterinary Sciences, The University of Agriculture, Peshawar, Pakistan 2Depatment of Livestock Management, Breeding and Genetics, College of Veterinary Science and Animal Husbandry, Abdul Wali Khan University, Mardan, Pakistan Article Information Received 16 March 2016 Revised 23 October 2016 Accepted 25 March 2017 Available online 28 December 2017


INTRODUCTION
I n the tropical and subtropical countries cross breeding was practiced during mid-seventies in order to improve herd productivity by cleaving the desired characteristics of higher dairy potential of Bos Taurus and enhanced resistance to heat stress and local diseases of Bos Indicus (Usman et al., 2014).The idea was to generate crossbred offsprings with early maturity, superior dairy characteristics and adaptability to harsh local environmental conditions (Usman et al., 2013;Sattar and Mirza, 2009).It has been suggested that over 50% crossbred juvenile bulls invested for semen collection had complications with quality of semen, cryo-tolerance and libido (Mukhopadhyay et al., 2010).Indigenous or pure breed bulls had comparatively higher freezing ability and fertility of spermatozoa than crossbred bulls (Gulia et al., 2010).Due to the poor semen (initial) quality nearly 50-55% of the ejaculates collected from the crossbred bulls, were unsuitable for freezing (Sudheer and Xavier, 2000).Various efforts have been made in order to isolate the etiology of poor quality semen production in crossbred bulls at seminological, andrological, genetic and probable ecological stages with varied success (Gulia et al., 2010).Due to the presence of some defective genes in bovine semen carrying special effect resulted in primary deformities.These deformities were comprised of acrosome shortcomings (ruffled, incomplete and knobbed), head deficiencies (decapitated, round head, abnormal head condensation, rolled head and nuclear crest), mid-piece defects (corkscrew, pseudodroplet and dag defects) and tail abnormalities (Chenoweth, 2005).It was reported that cross bred progeny is more prone to reproductive problems (Bitew and Prasad, 2011).One of the important factors of sub fertility in cattle is the use of substandard semen in artificial insemination (Gnoth et al., 2005).Generally the fertility is more vital in case of bull compared with cow, because one bull might be used for breeding nearly up to 40 cows through natural service or possibly hundreds thousands by means of artificial insemination.Though 20-40% bulls may have demoted fertility very few are entirely sterile (Coulter and Kastelic, 1999).A bull is considered to be selected for artificial insemination if its semen sample contains number of motile and live spermatozoa to reach the site of fertilization.Various tests have been conceived to assess the quality of semen in terms of viability, motility, concentration, acrosomal integrity and morphology (Amann and Hammerstedt, 1993;Pace et al., 1981).The present study was conducted to compare the semen quality of pure and crossbred cattle bulls under fresh and frozen conditions and to identify the best breed for the purpose of artificial insemination.

Fresh semen assessment
Selection of bulls Nine physically and andrologically healthy mature bulls of three different groups i.e.Sahiwal, Holstein-Friesian and crossbred (Sahiwal x Holstein-Friesian) at Semen Processing Unit in the Cattle Breeding and Dairy Farm Harichand were selected in order to compare the semen quality of pure and crossbred cattle bulls.The average age of bulls was 3 ± 0.14 years.All the experimental bulls were kept under the same shade and same feeding regime.Semen was collected from each bull early in the morning twice a week (Monday and Thursday).The experiments were continued for four weeks.

Semen collection
Semen was collected in a graduated tube from each experimental bull with a gap of 10 to 15 min using a teaser bull.The semen ejaculates having more than 60% motile spermatozoa, more than 1 mL semen volume and more than 0.5 billion/mL sperm concentration were considered for further semen quality tests (Ansari et al., 2011).For initial semen quality evaluation tests, the semen collected was immediately shifted to water bath at 34°C.

Semen evaluation
The following parameters were evaluated in fresh diluted semen of each bull.

Determination of seminal pH:
The pH was measured soon after dilution of fresh collected semen through pH meter.First of all, the semen sample was shacken properly and the normal color of the semen was checked.Later on, semen drops were taken and spread equally on pH paper.After 15 seconds the color of pH paper became uniform and impregnated, the color was then compared with the pH chart and the semen pH was decided considering 6.7 as the normal pH of semen (Haugen and Grotmol, 1998).

Determination of sperm individual motility and progressive motility:
A single drop of diluted fresh semen on pre warmed glass slide was taken and cover slip was placed over it.The individual motility percentage of sperm was visualized with a compound microscope at 400X at 34°C (Qureshi, 2011).Under microscopy, 100 spermatozoa were counted at five different fields.All those sperms, which showed any type of movement, were considered as motile spermatozoa while those sperm, which move in straight direction, were concluded as progressive motile.

Determination of sperm plasma membrane integrity:
Sperm plasma membrane integrity percentage was evaluated via hypo osmotic swelling test (Qureshi, 2011).A 0.1 mL diluted fresh semen was taken and mixed with 1 mL HOST solution which consists of 0.5 mL of D-Fructose (1.47%), sodium citrate (2.7%) and Eosin 0.5% (w/v).This mixed solution was incubated at 37°C for 40 min.After an incubation time, 5µL of sample with the help of micropipette was taken and kept on pre warmed glass slide.Cover slip was placed and visualized at 40X under compound microscope.A total of 100 spermatozoa were counted at five different fields.The HOST solution provided hypotonic media for spermatozoa, which caused coiling and swollen tail sperm due to different osmotic potential.Those sperm cell which showed swollen, coiled tail and unstained head represent intact plasma membrane integrity, while those with unswollen, uncoiled tail and pink head showed inactive sperm plasma membrane integrity.
Determination of dead spermatozoa: For dead sperm ratio, Eosin and Nigrosin stain was prepared, mixing 1% Eosin and 5% Nigrosin in 3% sodium citrate dehydrate solution (Zemjanis, 1970).Nigrosin provides a background for sperm cell visualization.Whereas, Eosin goes into the dead sperm cell membrane and highlightspink color for identification of dead sperm.A small drop of fresh diluted semen was taken on clean pre warmed slide and then added a comparatively large drop of stain, mixed it well enough by means of applicator stick for 30 seconds.A thin smear then was made, and air dried for 1 to 3 min.After drying, observed the slide under the compound microscope at 100X.The spermatozoa with unstained heads were considered live while those sperms which had stained heads or partial stained were considered dead spermatozoa.A total of 100 spermatozoa were counted at 5 different microscopic fields, and dead sperm percentage was calculated.
Determination of sperm acrosomal integrity: Trypanblue and Giemsa stain were conducted by the method of Kovacs and Foote (1992).In this protocol, trypan-blue was used to differentiate live and dead sperms and Giemsa stain was used for evaluation of sperm acrosomal integrity test.One drop of Trypan-blue (2.0%) was mixed with one drop of fresh diluted semen on a clean pre warmed slide, mixed by means of clean cover slip edge.The slide was then allowed to dry in air and formaldehyde-neutral red solution [86 ml IM HCl + 14 ml of Formaldehyde (Merck, Darmstadt, Germany) + 0.2g Neutral red (MP Biomedicals, Eschwege, Germany)] was used for 5 min for fixation.Slide was then washed with running distilled water and dipped in Giemsa stain (MP Biomedicals, Eschwege, Germany) (7.5%) for 4 h.After staining with Giemsa stain, slides were again rinsed with distilled water and kept in air to dry and cover slip was placed before covering with Balsam of Canada (Merck, Darmstadt, Germany).In each smear five different fields were evaluated to count a total of 100 sperms under phase contrast microscope at 1000X (LABOMED LX400).

Determination of sperm DNA integrity:
A 200 μL of TNE buffer was mixed with 20 μL diluted fresh semen at 37 °C to get the final concentration of 2 × 10 6 spermatozoa/ mL and clean glass slides were used for the preparation of three smears from each semen aliquot and air dried.The smears were dipped overnight in Carnoy's solution [methanol and glacial acetic acid (Merck, Darmstadt, Germany) in 3:1 ratio] for fixation.Slides were then allowed to dry in air and shifted to tampon solution (80 mM citric acid (Merck, Darmstadt, Germany) and 15mM Sodium phosphate (Sigma, NY, USA), pH 2.5) for 5 min.in water bath at 75 °C.After incubation in tampon solution, acridine orange (Sigma, NY, USA), pH 2.5) (0.2 mg/mL) was applied on slides.Slides were then covered with cover slip, while they were still soaked and evaluated under fluorescent microscope (LABOMED LX400).Acridine orange is fluorescent cationic dye having affinity for a nucleic acid and permeable to sperm cell interacts with single or double strand DNA by the process of intercalation or electrostatic attractions respectively.In each smear five different fields were evaluated to count a total of 100 sperms.Sperms DNA with green appearance indicated intact while sperms with disrupted DNA showed yellow green to red appearance (Evenson et al. 2002).

Post-thaw semen assessment Semen evaluation
The semen was collected from each bull and diluted with Tris-egg yolk extender and kept in water bath for 10 min.After extension the concentration of motile spermatozoa in extended semen was 50x10 6 per mL which was then forwarded to freezing process.The cooling was started from water bath temperature to 25°C and the changes were brought within 20 min.Further cooling started from room temperature to 4°C within 2 h and shifted to equilibration time for 4 h at 4°C (Leite et al., 2010).Straws filling were started with suction pump at cooled cabinet unit, kept on liquid nitrogen vapors for 10 min at 5 cm height and the straws were then plunged into liquid nitrogen and stored.After cryopreservation semen straws were thawed, incubated for 6 h and subjected to frozen semen quality tests (Ansari et al., 2011).After thawing the following tests were performed i.e., the seminal pH detection, individual and progressive motility, dead sperm, plasma membrane integrity, acrosomal integrity and DNA integrity for each bull in frozen semen.

Statistical analysis
Results of the study were presented as means ± SEM.To compare the fresh and frozen semen quality, sperm motility, progressive motility, membrane integrity, acrosomal integrity, semen pH, and DNA integrity, data were analyzed using two factorial designs.Duncan multiple range test was used to find the significance.A P value (P < 0.01) was statistically considered significant.

RESULTS
The results of the present study showed that membrane integrity, progressive motility, individual motility, acrosomal integrity and DNA integrity percentage were significantly high in fresh and frozen semen in Sahiwal breed compared with crossbred bulls (P < 0.01, Table I).Whereas, the seminal pH and dead sperm percentage were significantly high in the fresh and frozen semen of cross bred bulls compared with Sahiwal breed bulls (P < 0.01, Table II).

DISCUSSION
The current research was conducted at Government Cattle Breeding and Dairy Farm Harichand Charsadda and National Agriculture Research Council (NARC) Islamabad to find the comparison between semen traits of fresh and frozen semen in pure and crossbred bulls.Three different cattle breed bulls were selected in this research i.e.Sahiwal, Holstein-Friesian and cross-bred (Sahiwal x Holstein-Friesian), respectively.Each breed was tested for fresh and frozen semen condition to evaluate the semen quality.The data showed that sperm membrane integrity percentage of the semen was significantly (P < 0.01) high in pure breed (Sahiwal, Holstein-Friesian) compare with crossbred (Sahiwal x Holstein-Friesian) bull sperms.Sperm with intact plasma membrane plays a vital role to maintain and establish cellular homeostasis (Hammerstedt et al., 1990).Semen processing for future uses needs proper freezing and the spermatozoa are susceptible to freezing, which alters the sperm membrane activity.The degree of sperm membrane damage differs from any sort of changes in organization, membrane permeability, fluidity and lipid composition of sperm membrane bilayer (Thomas et al., 1998).It has been suggested that plasma membranes integrity in cross-bred bull sperms are affected more significantly than pure breeds during cryopreservation (Thippeswamy et al., 2014) which is in line with the results of the present study.Tyagi et al. (2003) demonstrated that reasons for culling the crossbred bulls from semen production stations were either their poor semen quality in fresh and frozen states or semen production was not suitable for further processing and cryopreservation and unacceptable for post thaw membrane integrity that satisfy the recommended standards for AI.
The research data showed that progressive motility percentage of the semen was significantly high in local Sahiwal bulls than crossbred, these results are in line with the study of Sudheer and Xavier (2000).The results of the present study are in agreement with various findings in the previous studies and found significant changes in progressive motility of sperm at different semen condition.The progressive motility can be defined as, those spermatozoa which move in a straight line direction in a microscopic field (Qureshi, 2011).Most damages to sperm occurs during semen freezing and thawing (ice crystallization and extra production of ROS) which deteriorate the plasma membrane and retarded the sperm movement (Aitken et al., 1998).In a study conducted on Frieswal crossbred (Friesian x Sahiwal) bulls, it was reported that 55% of the ejaculates produced by the crossbreed bulls were unsuitable for cryopreservation and the sperm progressive motility were unable to fertilize the ovum (Sudheer and Xavier, 2000).
In the present study, pure breed bull showed significantly higher individual motility than cross bred bull.We can define individual motility percentage as the spermatozoa with any movement in microscopic field called sperm individual motility (Qureshi, 2011).Sperm motility is the reliable tool that determines the fertility and viability of sperm (Janett et al., 2008).It is well known that ice crystal formation in mitochondria and axonemes during cryopreservation impairs sperm individual motility (Courtens et al., 1989).A large number of Frieswal (Holstein-Friesian x Sahiwal) bulls were rejected from semen station mostly due to the poor semen quality and post thaw motility (Mandal et al., 2012).
The sperm acrosomal integrity percentage was higher for pure breed bulls than cross-bred bull semen samples.The cranial end of spermatozoa nucleus is overlapped by acrosome and double layered thin membranous sac during the last stage of sperm formation.Intact acrosome cap of frozen thawed semen is directly related to the fertility of bulls because intact acrosome damages the zona-pellucida by means of hydrolytic enzyme, during the process of fertilization (Saacke and White, 1972).Prior to fertilization, sperm undergoes capacitation.Cryopreservation induces disturbance in premature capacitation, resulting in low fertility of frozen semen (Watson, 2000).In crossbred bulls, the morphology of abnormal spermatozoa might possess damaged 50%-55% acrosome and at least of the ejaculates were unsuitable for freezing due to the poor initial semen quality (Sudheer and Xavier, 2000).
Normal pH of bull semen is 6.7.Storage of semen at room temperature may decrease the pH due to production of lactic acid from fructose (Qureshi, 2011).Semen pH may become alkaline when dead sperm percentage is high (Qureshi, 2011).The maximum average pH was obtained from crossbred bull semen after thawing the semen (Hossain et al., 2012).The presented data is supported by the above findings as for crossbred bull semen, having large number of dead sperm which results in higher pH as compared to the pure bred bull frozen semen samples.
The present data show that DNA integrity in fresh and frozen semen showed significantly higher value for pure breeds (Sahiwal, Holstein-Friesian) than crossbred (Sahiwal x Holstein-Friesian) bull semen.Sperm DNA is the molecular basis of heredity, constructed of a double helix held together by hydrogen bonding (Balhorn et al., 2000).DNA starts division and reunion during spermatogenesis of sperm in meiotic cell division (Rajendera et al., 2011).The different steps of cryopreservation i.e. dilution, cooling, equilibration time and thawing of frozen semen enhance production of reactive oxygen species (Baumber et al., 2005).DNA integrity is highly susceptible to increase production of ROS, which occurs at plasma membrane (Kankofer et al., 2005).The excessive ROS production is the major cause of damage to the sperm DNA integrity and this damage is observed comparatively more in crossbred bulls than pure (Garg et al., 2008).Some outdoor factors that also influence the sperm DNA structure include types of semen extenders, dilution methods, duration of incubation and thawing of cryopreserved semen (Hammadeh et al., 1999).Dead sperm percentage value was significantly high for cross-bred bulls followed by pure breed bull semen samples.Sperm viability is one of the most important requirements of fertile semen (Hossain et al., 2012).Both freezing and thawing implicate tremendous alterations in cell water volume, which result in considerable mechanical stress on the membrane and consequently reduce sperm viability (Hammerstedt et al., 1990).The sperm plasma membrane is the primary site of damage induced by cryopreservation, which affects the viability of sperm (Hammerstedt et al., 1990).Average motility of sperm before and after freezing varied, which indicate that freezing of semen reduced sperm motility and increased dead ratio irrespective of breed.

CONCLUSION
Semen from cross-bred bulls showed more susceptibility to damage in fresh and cryopreservation than pure breed bull semen.Among the different cattle breed bulls Sahiwal showed the best semen quality traits.

Statement of conflict of interest
Authors have declared no conflict of interest.