Addition of L-Cysteine and Vitamin E to Semen Diluent Enhances Freeze-thawed Spermatozoa Characteristics in Crossbred Cattle Bulls under Subtropical Environment

Ibrar Muhammad Khan1, Dezhi Xu1, Zubing Cao1, Hongyu Liu1, Adnan Khan2, Sajid Ur Rahman3, Jam Zaheer Ahmed4, Muhammad Akmal Raheem5 and Yunhai Zhang1* 1Anhui Provincial Laboratory of Local Livestock and Poultry Genetical Resource Conservation and Breeding, College of Animal Science and Technology, Anhui Agricultural University, Hefei, Anhui, China, 230036 2Department of Animal Genetics, Breeding and Reproduction College of Animal Science and Technology, China Agricultural University, Beijing, China 3Key Laboratory of Animal Parasitology of Ministry of Agriculture, Laboratory of Quality and Safety Risk Assessment for Animal Products on Biohazards (Shanghai) of Ministry of Agricultural Sciences, Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Shanghai 200241, China 4State Key Laboratory for Conservation and Utilization of Subtropical AgroBioresources, Guangxi University, Nanning, Guangxi, China 5Key Laboratory of Veterinary Pathobiology and Disease Control, College of Animal Science and Technology, Anhui Agricultural University, Hefei, China Article Information Received 06 October 2019 Revised 29 December 2019 Accepted 28 January 2020 Available online 26 August 2020


INTRODUCTION
T he idea behind cross breeding is to generate crossbred offspring with early maturity, superior dairy characteristics and adaptability to harsh local environmental condition. However, little attention has been devoted to improving the quality of breeding programs (Sattar and Mirza, 2009). Over 50% of crossbred juvenile bulls used for semen collection were reported to have complications related to the quality of semen, cryo-biology and libido O n l i n e

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use of substandard semen in artificial insemination (AI) (Gnoth et al., 2005). Sub-fertility may occur as a result of disorders in semen production including reduced or loss of sperm motility (Dirami et al., 2013). Cryopreservation is the key technique for artificial insemination and providing a platform to distribute the elite genetic material worldwide but the freezing-thawing cycle can have detrimental effect on sperm biology, causing damage and modifications that eventually lead to the death of sperm cell, thereby reducing fertility (Trachootham et al., 2009). Cryopreservation can generate reactive oxygen species (ROS) which interact with sperm cell plasma membrane leading to trans-omics disturbance and suffer the fertility. When ROS exceed the normal level and spermatozoa defence mechanisms are decreased not only the plasma structure but also DNA can be damaged. This damage can reduce spermatozoa fertility and the capability to develop a healthy embryo (Pons-Rejraji et al., 2009). Another study found that low ROS production is positively correlated with spermatozoa motility whereas high ROS production was negatively correlated with sperm motility. This ROS-induced decline in spermatozoa motility, is primarily attributed to decreased ATP production (Armstrong et al., 1999).
Previous studies have found that the antioxidant property of vitamin E can improve the freeze-thawed traits of spermatozoa and contribute to the management of male infertility (Keskes-Ammar et al., 2003). Vitamin E and beta-carotene have been shown to have the antioxidant property that support cells against ROS damage (Almbro et al., 2011). Vitamin E is considered to be the most active agent in the antioxidant system of spermatozoa, where it protects the cell membrane against attack from ROS and lipid peroxidation (LPO) (Yousef et al., 2003). The administration of antioxidant agents to semen diluent has been improving the indicators of spermatozoa motion (e.g. spermatozoa longevity, individual motility, progressive motility and viability) in Ghezel rams (Aminipour et al., 2013). However, cattle bull semen has natural guard system against the ROS and, that are considered insufficient, when the spermatozoa going through the critical phase of freezethawed imposed stress . Therefore, it is essential to strengthen the semen diluent with appropriate antioxidant supplements to reduce ROS damages durring the freeze-thawing of bull semen. Cysteine is another active agent, also known to act as an intracellular antioxidant and guard's cells against oxidative stress and damage (Meister and Anderssson, 1983). L-cysteine comprises an important thiol group that works as a non-enzymatic antioxidant and easily penetrates into sperm cells. The addition of cysteine to semen diluent has been reported to improve the freeze-thawed motility and viability of boar (Kaeoket, 2010), buck (Kulaksiz and Daskin, 2010) and bull semen (Sariözkan et al., 2009).
This study assessed the effect of L-cysteine and vitamin E administration to semen diluent on freeze-thawed spermatozoa characteristics. The ideal concentrations of antioxidant supplements for the cryopreservation media of crossbred bull semen was also determined that resulting in the good post-thawed semen characteristics. We perceived that addition of L-cysteine and vitamin E to semen diluent would improve the freeze-thawed semen qualities (e.g., spermatozoa motility, membrane functionality, acrosomal integrity, DNA integrity, and spermatozoa viability ratio and mitochondrial membrane potential) of crossbred bull.

Animals and rearing system
Six physically and andrologically healthy mature bulls of two crossbred groups, (Sahiwal × Holstein-Friesian) and (Achai × Jersey), were selected from the National Agriculture Research Center (NARC) in Islamabad. The average age of the bulls were 3.0 ± 0.14 years. All experimental bulls were reared under the same feeding regime and housing facilities. Seasonal green fodder adlibitum and formulated feed (e.g., maize gluten 30 of 15%, cotton seed cake 18%, maize grain broken 14%, canola meal 10%, molasses 16%, wheat bran 25%, and mineral mixture 2%) were being offered.

Semen collection
According to standard artificial insemination procedure, total of sixteen ejaculates were collected from each bull. The following criteria were considered for collected ejaculates: volume of 2-6 mL, concentration ≥ 60 × 10 6 spermatozoa mL _1 and total motility of 70% for conducting the post-thaw experiments . The volume of collected semen was measured while using a graduated tube and the initial spermatozoa total motility was determined microscopically (magnification 400X; Olympus BX20, Tokyo, Japan). The fresh spermatozoa concentration was evaluated using a digital photometer (L'Aiglon; IMV, Cedex, France) and pooled ejaculates were used for further assessment.

Addition of L-cystiene and vitamin E in semen diluent
The stock diluent comprised of 1.56% citric acid (Fisher Scientific, UK), 3.0% tris-(hydroxymethyl)aminomethane (Research Organics, USA), 0.2% glucose (Scharlau, Spain), 7% glycerol (Riedel-deHaen, Germany), 20% egg yolk in distilled water, at pH 6.8 and standard osmotic pressure of 320 mOsmol kg-1 for cattle bulls was used (Beheshti et al., 2011). The semen samples 60 x 10 6 O n l i n e F i r s t A r t i c l e spermatozoa / mL concentration were used for dilution and then divided into seven different experimental fractions. The 1st, 2 nd and 3 rd fractions were diluted containing Cysteine (C8786; Sigma-Aldrich Co., St. Louis, MO, USA) at concentrations of 5 mmol (L-cysteine-1), 7.5 mmol (L-cysteine-2) and 10 mmol (L-cysteine-3); the 4 th , 5 th and 6 th fractions were diluted containing Vitamin E (Sigma-Aldrich Co., St. Louis, MO, USA) at concentrations of 1.2 mmol (vitamin-E-1), 2.4 mmol (vitamin-E-2) and 4.8 mmol (vitamin-E-3) whereas the 7th (control fraction) was without any addition of additives to the diluent. Vitamin E was dissolved in absolute ethanol of (0.05% v/v) to prepare a stock solution of 10 mM before it was added to the diluent (Motemani et al., 2017).

Cryopreservation
All diluted samples were cooled at 4 °C over 2 h in cooled cabinet and after cooling the samples were exposed to equilibration time (e.g., total refrigeration time 4 h at 4 °C) (Leite et al., 2010). At the end of cooling and equilibration period the extended samples were added to 0.25 mL French straws and sealed with a filling and sealing device (IMV, L'Aigle, France). The semen straws were cryo-preserved through programmable bio-freezer (Minitub, Tiefenbach, Germany) at -20°C/min to -100°C and at -10°C/min from -100°C to -140°C and finally plunged into liquid nitrogen. After a week, the frozen straws were thawed for freeze-thawed assessment.

Post-thawed semen evaluation
The following parameters were evaluated in the freeze-thawed semen of bulls whereas all the parameters were tested for three times consecutively.

Spermatozoa total motility and progressive motility
The spermatozoa total motility and progressive motility parameters were assessed by computer assisted sperm motion analysis (CASA, Version 5.1; Microptic Barcelona, Spain). A total of 10 µL of freeze-thawed semen was placed in pre-warmed Makler chamber (Sperm Meter; Sperm Processor Pvt. Ltd, Aurangabad, India) and examined. We randomly selected three microscopic fields each scanned five times. The mean of these fifteen scans was used for spermatozoa motility analysis.

Spermatozoa membrane functionality (Hypo-osmotic swelling test)
Spermatozoa plasma membrane functionality was determined via hypo-osmotic swelling tests (Pons-Rejraji et al., 2009). A total of 0.1 mL freeze-thawed semen was mixed with 1 mL HOST solution consisting of 0.5 mL D-fructose (1.47%), sodium citrate (2.7%) and eosin 0.5% (w/v). Following incubation at 37°C for 40 min, 5 µL of sample was removed with micropipette, transferred to a pre-warmed glass slide while covered with cover slip and placed under the compound microscope at 40X for observation. A total of 200 spermatozoa were counted in five different fields for each sample. The HOST solution provided hypotonic media for the spermatozoa, resulting in coiled and swollen tails due to the different osmotic potential. Intact plasma membrane functionality was indicated by swollen, coiled tails and unstained heads, whereas un-swollen, uncoiled tails, and pink heads indicated disruptive spermatozoa membrane functionality.

Spermatozoa viability
To determine the ratio of viable spermatozoa, eosin (1%) and nigrosin (5%) stain was mixed in 3% sodium citrate dehydrates solution. Nigrosin stain provides a background for spermatozoa visualization while eosin enters into the dead spermatozoa membrane and produces a pink color, allowed dead spermatozoa identification. A small drop of freeze-thawed semen was placed on clean pre-warmed slide. A comparatively large drop of stain was added and mixed for 30 s using an applicator stick. A thin smear was then made and air dried for 1 to 3 min. After drying, the slide was observed under the compound microscope at 100X. The spermatozoa with unstained heads were considered live while those with stained or partially stained heads were considered dead. For each sample, the percentage of spermatozoa viability was calculated based on a total of 200 spermatozoa counted in 5 different microscopic fields.

Spermatozoa acrosomal integrity
Trypan-blue and giemsa staining were conducted for spermatozoa acrosomal integrity in bull (Kovacs and Foote, 1992). In this protocol, trypan-blue was used to differentiate live and dead sperms, and giemsa stain was used to evaluate sperm acrosomal integrity. One drop of trypan-blue (2.0%) was mixed with one drop of freezethawed semen on a clean pre-warmed slide and mixed using the edge of a clean cover slip. After drying in air, the slide was fixated with formaldehyde-neutral red solution [86 mL IM HCl + 14 mL formaldehyde (Merck, Darmstadt, Germany) + 0.2g neutral red (MP Biomedicals, Eschwege, Germany)] for 5 min. Subsequently the slide was washed with distilled water and dipped in (7.5) giemsa stain (MP Biomedicals, Eschwege, Germany) for 4 h. After staining with giemsa stain, the slide was again rinsed with distilled water and dried in air. After covering with cover slip, the slide was covered with Canada balsam (Merck, Darmstadt, Germany). For each smear, five different fields were evaluated under a phase-contrast microscope at 1000X

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(LABOMED LX400), in resulting analysis of total 200 spermatozoa. As trypan-blue stain is permeable to none viable sperm and reflect blue appearance, whilst color of viable spermatozoa with intact membrane remained unchanged. In contrast, spermatozoa with intact acrosomes are permeable to giemsa stain, which turns the acrosomal part purple (Tartaglione and Ritta, 2004).
Spermatozoa DNA integrity TNE buffer (200 μL) was mixed with 20 μL of freezethawed semen at 37 °C to obtain a final concentration of 2 × 10 6 spermatozoa / mL. Clean glass slides were used to prepare three smears from each semen aliquot. After drying in air, the smears were dipped overnight in Carnoy's solution [methanol and glacial acetic acid (Merck, Darmstadt, Germany) in a 3:1 ratio] for fixation. The slides were then allowed to dry in air and shifted to tampon solution [80 mMol citric acid (Merck, Darmstadt, Germany)] and 15 mmol sodium phosphate (Sigma, NY, USA), pH 2.5) for 5 min in a water bath at 75 °C. After incubation in tampon solution acridine orange (0.2 mg / mL, pH 2.5; Sigma, NY, USA) was applied to the slides. The slides were then covered with cover slips while they were still soaking and evaluated under fluorescent microscope (LABOMED LX400). Acridine orange is a fluorescent cationic dye with affinity for nucleic acids and permeable to sperm cells. Acridine orange interacts with single or double-strand DNA via intercalation or electrostatic attraction respectively. For each smear, five different fields were evaluated to count a total of 200 sperms. Intact sperm DNA with indicated by a green appearance while disrupted DNA appeared yellowish green to red (Evenson et al., 2002).
Mitochondria membrane potential JC-1 is a lipophilic cationic dye and sensitive marker for mitochondrial membrane potential (MMP) (T4069; Sigma -Aldrich Co.), was used for assessment of sperm MMP. In spermatozoa with high MMP, thereby the JC-1 marker forms multiplexes, known as J-aggregates, and releases orange fluorescence, while spermatozoa with low MMP then the JC-1 leftovers in monomeric form and releases green fluorescence. The thawed ejaculate samples were centrifuged for 5 min at 500(x) g and supernatant fluid was then discarded. The residual spermatozoa were diluted in phosphate buffer saline at a concentration of (1 x 10 6 ) spermatozoa per mL, afterward 1 mL of the suspension was stained with 1 µL of JC-1 stock solution (200 µMol dissolved in DMSO) and incubated at 38°C for 40 min, and eventually examined by flow cytometry. Red and orange fluorescence of JC-1 dye was noticed (530 nm) with FL1 and (585 nm) FL2 detectors, correspondingly (Minervini et al., 2013).

Statistical analysis
The obtained data were statistically analyzed using SPSS version 16, SPSS Inc., Chicago, IL, USA. The Kolmogorov Smirnov test was employed to test the normal distribution of all the collected data. The results of this study are presented as (Mean ± SEM). The total motility, sperm viability, membrane functionality, acrosomal integrity, DNA integrity and mitochondria membrane activity percentage of freeze-thawed spermatozoa of crossbred bulls were comparing between the different experimental diluent groups using one-way analysis of variance (ANOVA). Duncan multiple range test to determine significant difference; the value of (P <0.05) was considered statistically significant.

RESULTS AND DISCUSSION
The impact of L-cysteine and vitamin E on freezethawed sperm motility in both of crossbred bulls are demonstrated in Table I, Figures 1 and 2. Addition of antioxidants (L-cysteine and vitamin E) to semen freezing diluent with 7.5 mmol of L-cysteine and 4.8 mmol of vitamin E improved motility of freeze-thawed spermatozoa as compared to the control group (P < 0.05) followed by 5 mmol of L-cysteine and 2.4 mmol of Vitamin E groups. Similarly, 7.5 mmol L-cysteine, and 4.8 mmol of vitamin E groups are yielded highest progressive motility and that was statistically significant as compared to control group (P < 0.05). The study design consisted of two crossbred groups e.g. Sahiwal × Holstein-Friesian and Achai × Jersey while parallel with different concentrations of both L-cysteine and vitamin E supplements. Both of crossbred groups were tested under different concentrations of L-cysteine and vitamin E to evaluate freeze-thawed spermatozoa traits. The results showed that motility parameters of freeze-thawed spermatozoa were significantly higher (P < 0.05) in group of L-cysteine-2 and vitamin-E-3 for both types of crossbred bulls. Spermatozoa motility is crucial to cross the female reproductive tract and hurdles around the oocyte for succeeding fertilization (Bucak et al., 2012). As formerly described, spermatozoa motility and viability are usually affected features during freezing-thawing cycle and hence, these are the crucial causes for reduced fertility (Li et al., 2010). Data about the sperm motility in our study are wholly in conformity with different studies in which addition of L-cysteine and vitamin E augmented motility of freeze-thawed bull (Amirat-Briand et al., 2009), ram  and buck spermatozoa (Kulaksiz and Daskin, 2010).

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Antioxidant Agents Enhances Freeze-thawed Semen Qualities    Previous reports designated that addition of L-cysteine with concentration of 5 and 7.5 mmol in buffalo semen freezing extender significantly improved the motility of freezethawed spermatozoa (Beheshti et al., 2011). The vitamin E is measured to be the main constituent of antioxidant system for spermatozoa (Lenzi et al., 1996). In latest studies, the supplementation of vitamin E into freezing diluents has been amended the freeze-thawed spermatozoa functions of ram , boar (Breininger et al., 2005) and tom (Thuwanut et al., 2008). Likewise, the addition of vitamin E into semen freezing extender resulting in optimistic effect on spermatozoa motility, integrity of membrane, DNA and MMP (Jeong et al., 2009). As shown in Table I and Figures 1 and 2 supplementation of crossbred bulls freezing diluent with 7.5 mmol of L-cysteine and 4.8 mmol of vitamin E are significantly improved membrane functionality of spermatozoa along with significant increase in acrosomal integrity as compared to control group (P < 0.05) and followed by the other groups. In this study, membrane functionality of freeze-thawed spermatozoa was significantly higher (P < 0.05) in groups of L-cysteine -2 and vitamin-E-3 compared with the control group. Plasma membrane is accountable for the conservation of cellular homeostasis and thereby the plasma membrane functionality applies a vital role on spermatozoa survival inside the female reproductive tract and, on conservation of spermatozoa fertilizing capability (Andrade et al., 2007). Semen collection and processing for future uses requires proper freezing methods. Spermatozoa are prone to freezing, which is altered sperm membrane activity and the degree of spermatozoa membrane damages differs based on organization, membrane permeability, fluidity and the lipid composition of sperm membrane bilayer (Motemani et al., 2017). Plasma membrane integrity in crossbred bull spermatozoa has been suggested to be significantly affected by cryopreservation compared to pure breeds (Hammerstedt et al., 1990). Lipid peroxidation (LPO) of plasma membrane disturbs membrane construction through ROS production (Thomas et al., 1998), while leading to deterioration in critical sperm properties, including membrane activity, motility and fertilization ability (Thippeswamy et al., 2014). It has been recommended that vitamin E delivers a biological stability to the spermatozoa plasma membrane while as the increase nutritional intake of vitamin E content in resulting the semen was found less prone to LPO (Surai et al., 2000;El-Sheshtawy et al., 2008). El-Sheshtawy et al. (2008) found that adding L-cysteine (7.5 mmol) to tris-citric acid diluent increased the percent of intact membranes spermatozoa in Egyptian buffalo after cryopreservation. Likewise, treating tris-based diluent with L-cysteine (7.5 and 10 mmol) resulted in enhanced percentages of sperm with efficient plasma membranes in bulls (Sariözkan et al., 2009) and rams , in agreement with our findings.
In this conducted research, spermatozoa acrosomal integrity in both types of crossbred bulls was significantly higher (P < 0.05) in groups L-c-2 and vit-E-3 compared with control group. The percentage of intact acrosomes is positively related to fertility in freeze-thawed bull spermatozoa (Hafez, 2004). The intactness of acrosomes and their enzyme (acrosin and hyaluronidase) activities are indispensable for acrosomal reaction and spermatozoaoocyte fusion (Sudheer and Xavier, 2000). The structural and functional integrity of the sperm acrosome is considered necessary to attain the high fertility however cryopreservation can damage the acrosomal layer, which diminishes the ability of spermatozoa to penetrate the zona pellucida (Yanagimachi, 1994). Some studies have suggested that vitamin E protects against LPO caused by ROS in bull and boar Breininger et al., 2005). In this study the groups treated with vitamin E had larger percentages of sperm with intact acrosome membranes compared to the control group. In addition, L-Cysteine has the aptitude to rapidly pass into the cells, where it is altered to taurine. The taurine is united with fatty acids in membrane and altered into acyl-taurine, improving the surfactant properties and osmoregulation of the spermatozoa membrane (Esteves et al., 2007;Vazquez and Roldan, 1997).
The percentage of spermatozoa with high viability and increased DNA integrity in both of crossbred bulls were found when 7.5 mmol of L-cysteine and 4.8 mmol of vitamin E were added in freezing diluent (P < 0.05) compare to control group. Similarly, high MMP was observed when the freezing diluent treated with 7.5 mmol of L-cysteine and 4.8 mmol of vitamin E as compare to control group (P < 0.05) followed by the other treated groups as shown in Table I, Figures 1 and 2. In this study the DNA integrity of freeze-thawed spermatozoa was significantly higher (P < 0.05) in groups L-c-2 and vit-E-3 in both types of crossbred bulls. Cryopreservation method was found to damage the DNA integrity of buffalo spermatozoa, which affects the sperms functional credits and successful fetal development (Kumar et al., 2011) and, can also decrease the antioxidant potential of sperm. Antioxidants contribute in protecting DNA integrity during freeze-thawing  by decreasing oxidative stress. Freeze-thawing induces adverse events such as ROS production and apoptosis-like changes; these events disturb the packaging of the sperm chromatin, in result a loss of DNA integrity (Topraggaleh et al., 2014). L-cysteine is sulfur-containing amino acids that are naturally found in seminal plasma and sperm nucleic acid. L-cysteine preserves DNA integrity and acts O n l i n e

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Antioxidant Agents Enhances Freeze-thawed Semen Qualities 7 as an antioxidant directly and /or indirectly to defend cells against ROS (El-Sheshtawy et al., 2008;O'Flaherty et al., 1997), in agreement with the findings of the study. As vitamin E is a known inhibitor of LPO in cell membranes and acts as scavenger of ROS, thereby reducing oxidative damage during freeze-thawing of bull semen (Saacke and White, 1972). Cryopreservation induced structural damages in mitochondria which altered the biochemical processes involved in ATP production, ultimately resulting in reduced sperm viability and motility (Figueroa et al., 2017). Mitochondria membrane is the key site of ROS production due to oxidative phosphorylation (Thuwanut et al., 2011). Kadirvel et al. (2009) found a negative relationship between mitochondrial membrane activity and ROS in freeze-thawed buffalo spermatozoa. Direct correlation exists among the MMP, sperm motility and fertilization capacity of freeuze-thawed spermatozoa. This relationship confirmed that cessation or activation of motility and successful fertilization depends on the availability of ATP in the spermatozoa (Fraser and Strzezek, 2007). The significant growth in MMP of freeze-thawed spermatozoa supplemented with L-cysteine and vitamin E could be due to the high antioxidant potential of L-cysteine and vitamin E to eliminate intracellular ROS and afterward protect mitochondria membrane activity.
The percentage of viable spermatozoa was significantly higher (P < 0.05) in groups L-cysteine -2 and vitamin-E-3 compared to control group. Spermatozoa viability and motility are the most essential requirements for fertile spermatozoa (Hossain et al., 2012). Freeze-thawing cycle results in substantial alterations in spermatozoa water content, creating considerable mechanical stress on the membrane and reducing spermatozoa viability (Hammerstedt et al., 1990). The addition of L-cysteine (7.5 mmol) to tris-citric acid diluent was found to improvement the percentage of viable ovine spermatozoa (Bucak et al., 2008). Under field conditions, decrease in viable spermatozoa after freeze-thawing significantly reduces the fertility of bovine semen (Andrabi et al., 2006). In similar studies on ovine semen the addition of L-cysteine to semen extender resulted in higher percentage of live spermatozoa in association with increased catalase activity (Thomas et al., 1998;El-Sheshtawy et al., 2008). Our results show that vitamin E exhibits cryo-protective effects through its ability to reduce ROS accumulation. Our findings agree with several studies in which the addition of vitamin E to diluents enhanced the rate of motility and viability of spermatozoa (O'Flaherty et al., 1997;Hafez, 2004).

CONCLUSIONS
Addition of L-cysteine or vitamin E supplements at concentrations of 7.5 and 4.8 mmol to semen diluents respectively, providing more strength to MMP and afterward elevated the spermatozoa freeze-thawed motility parameters in both crossbred bulls compared with control. In addition our study confirmed that adding 7.5 mmol L-cysteine and 4.8 mmol of vitamin E in freezing diluents enhanced sperm membrane functionality and DNA integrity. Therefore, these supplements can be suggested as an additional component for freezing diluent while in semen preservation of crossbred bulls. Further studies are recommended, to assess in vivo and in vitro fertility rates of freeze-thawed crossbred semen preserved with L-cysteine and vitamin E.