Post Thawed Quality of Tharparkar Bull Semen Extended in Bioxcell TM and Tris-Based Egg Yolk Extenders Supplemented with Vitamin E (α-Tocopherol)

The study was designed to determine the effect of Vitamin E (α-tocopherol) supplimentation into BIOXcell TM and Tris based egg yolk extender on post-thawed quality of Tharparkar


INTRODUCTION
L ivestock plays a pivotal role in the social and economic development of Pakistan. Livestock is the most important sector of agriculture. It contributes up to 60.1 % in the agricultural value added and about 11.7 % to the gross domestic product (GDP) of Pakistan during 2020-21, Moreover total livestock population in Pakistan O n l i n e

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is 213.1 million heads including cattle 51.5 million (GOP, 2020-21). Cattle belong to the family Bovidae. Cattle were originated from Bostaurus or humpless cattle (Europe) and Bosindicus or humped cattle (Indo-Pakistan), The cattle breeds of Pakistan are Red Sindhi, Bhagnari, Sahiwal, Dhanni, Kankrej, Rojhan, Lohani, Cholistani, Dajal and Tharparkar (Farooq et al., 2013).Tharparkar is a lyrehorned breed that originated from the Thar Desert (Chand, 2011;Memon et al., 2022). It is one of the main breeds which is found in the Thar Desert mainly located in the India-Pakistan border,It is considered a dual purpose breed utilized as a draught and milking animal (Godara et al., 2015). Meanwhile, it has been blessed with the capabilities of heat tolerant, tick resistant, disease-resistant and a stable aboriginal indigenous breed.Tharparkar breed is producing approximately 5-10 L of milk per day and round about 1135-2000 L of milk per lactation . Extenders are used for conservation, preservation and extension of semen. Moreover, extenders are also used to prevent different types of damages while processing, storage and shipping of semen. During the last 40 years, different extenders have been used for the evaluation of spermatozoa during cooling and post-thawing. Egg yolk-based semen diluents are readily used for the cryopreservation of semen (Apu et al., 2012;Emamverdi et al., 2014). Different components are combined for the preparation of semen extenders, such that they own all properties which have capabilities to protect the life of spermatozoa during extension at ambient environment and cryopreservation. It must be isotonic (maintain osmotic pressure) (280-310 mOsm/kg), maintain pH, cold shock defense, act as a source of energy (spermatozoa metabolism), antimicrobial, protect during cooled and post thawing and able to preserve sperm fertility for the long time duration (Raheja et al., 2018). Tris-based egg yolk extender is used for the extension of semen though it is a mixture of a substance having cryoprotectant properties. With long time storage, it shows qualities of a great stabilizing agent with constant results (Apu et al., 2012;Emamverdi et al., 2014).
BIOXcell TM is a commercial lecithin based extender. However, it has been used in many studies on exotic breeds but in Tharparkar cattle it's merely not used. Moreover, it has positive effects on the cryopreservation of semen so there was a dire need to study the effect of BIOXcell TM extender in Tharparkar cattle bull semen. BIOXcell TM has merits in case of lower sanitary risks, its chemically defined and ready to use meanwhile its commercially available (Akhter et al., 2010). The process of cryopreservations also leads to various intracellular changes which results in the production of reactive oxygen species (ROS) that are indirect cause of reduced reproductive capacity, DNA damage, increased membrane permeability. ROS damages plasma membranes and DNA molecules in the sperm and other cells. High levels of superoxide ions, peroxynitrates hydrogen peroxide, harm the components of cells such as membrane lipids, organelles, proteins and DNA. To overcome ROS antioxidants are added, Vitamin E is the main antioxidant sited within the biological membranes that perform a key role in defending from lipid peroxidation. α-tocopherol breaks the chain reactions of lipid peroxidation but the mechanism of donation of a hydrogen atom from its phenolic hydroxyl group to lipid peroxyl radical resulting in the creation of stable lipid hydroperoxide and unreactive tocopheroxyl radicals. The antioxidant property of vitamin E can improve the postthawed traits of spermatozoa and could improve male fertility. Vitamin E has the antioxidant property that supports sperm against ROS damage (Almbro et al., 2011). The addition of antioxidant agents in semen extenders improves sperm longevity, individual motility, progressive motility and viability (Aminipour et al., 2013). Though cattle bull semen has a natural defense system against the ROS but that is not enough to prevent sperm against changes in decrease and increase in the temperature during the process of cryopreservation (Nichi et al., 2006;Khan et al., 2021). Artificial insemination and estrus synchronization are the techniques that provide opportunities to control the estrus cycle and reproductive management in bovines (Senger, 2005;Lijalem et al., 2015). For estrus synchronization, it is necessary to have cyclic cows with proper nutrition, good body condition scores (BCS) and high-quality semen (Lamb, 2012;Gizaw and Dima, 2016). There were several studies available on crypreservation but few were reported on indigenous Tharparkar cattle bull semen. Therefore, this study was designed with a hypothesis that supplementation of vitamin E (α-tocopherol) into BIOXcell TM and Trisbased egg yolk extender would enhance the post-thawed quality of Tharparkar cattle bull semen.

MATERIALS AND METHODS
The study was carried out at Department of Animal Reproduction, Faculty of Animal Husbandry and Veterinary Sciences, Sindh Agricultural University Tandojam. In this study four fertile Tharparkar bulls (A, B, C and D) having age of 4-5 years were used. A total of 52 (n=13) ejaculates were collected with the help of artificial vagina, After the collection macroscopic (color, volume and pH) and microscopic (motility, morphology, viability, concentration and membrane integrity) parameters were observed. The samples having motility, morphology, viability and membrane integrity ≥ 70% were pooled and processed. Pooled semen samples were divided into four groups and diluted with Tris, Tris +Vitamin E(αtocopherol) 0.02 Mm/ml and BIOXcell TM , BIOXcell TM + Vitamin E(α-tocopherol) 0.02 Mm/ml.

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The volume of semen was observed through visual examination with the help of a graduated tube. Visual examination was done for judgment of colour however semen was categorized as milky, creamy white and translucent (Kaka et al., 2012). pH of semen was determined by using a digital pH meter (Hanna Company, Instrument number #HI98107, software version v1.01, serial number # HA02122313, Made in Romania).
The wave motion was assessed on a clean warm dry slide by putting a drop of undiluted semen under low power magnification (10X) with a phase-contrast microscope (Nikon, Germany). The wave sample was recorded and classified as described by Rehman (2012).
For the assessment of motility, the semen sample was diluted in normal saline at 1:100. After one drop of diluted sample was taken and put on a pre-warmed slide by applying a cover slip on it. While using (20 xs) underlow magnification Motility was observed. Motility percentage was taken by selecting 100 spermatozoa moving randomly in a straight forward direction. Spermatozoa that were stirring in a backward direction or circle were not counted moreover results were expressed in motility percentage. The sample with 70% motility was processed for further assessment.
The concentration of sperm was assessed with the help of haemocytometer as described by Kaka et al. (2012). As per the standard staining procedure of sperm, morphology was determined as described by (Kaka et al., 2012) and The viability of sperms was recorded as described by Kaka et al. (2012). Hypo osmotic swelling (HOST) test method was used to determine the membrane integrity of fresh semen samples as described by (Kaka et al., 2012).
The diluted semen were packed into straws (0.25 ml). Moreover, straws were classified as red for BIOXcell TM (control group), white for BIOXcell TM (vitamin 0.2mM), yellow for tris-based egg yolk extender (control group) and blue for tris-based egg yolk extender (vitamin 0.2mM). Semen straws were filled with a manual suction machine. The semen straws were sealed with polyvinyl pyrrolidone powder (PVP). The process of filling and sealing were performed in the cold cabinet to maintain a similar temperature and prevent shock. The equilibration period was completed within 2 h at 5 o C.
Freezing was carried out by holding the straws in liquid nitrogen vapors 5 cm above the surface of liquid nitrogen for 6 min as described by Kaka et al. (2012). Then the straws were plunged into liquid nitrogen (-196 o C). The frozen semen were stored in liquid nitrogen at least for 24 h and then assessed for post-thaw qualities parameters i.e. motility, morphology, viability and membrane integrity were followed (Rasul et al., 2000;Kaka et al., 2012).
The collected data were subsequently subjected to a one-way analysis of variance (ANOVA) using Statistics (2006) and LSD was used to determine the difference among means of different groups.

RESULTS AND DISCUSSION
The results of macroscopic parameters i.e. volume, color, pH and microscopic parameters i.e. wave motion, motility, concentration, morphology, viability and membrane integrity of freshly collected semen from bulls A, B, C and D are depicted in Table I. Results showed a slightly highest (Mean % ± SEM) values of semen from bull D. However, no significant difference (P>0.05) were recorded amongst bulls.   Table II represent the (Mean±SEM) post-thaw assessment of semen quality parameters i.e. motility, morphology, viability and membrane integrity in BIOXcell -TM and tris based egg yolk extender supplemented with and without vitamin E (α-tocopherol). Improved quality parameters were observed in BIOXcell TM supplemented with vitamin E (α-tocopherol). Moreover, significant difference (P<0.05) was observed among all groups. Table III presents the percentage of conception rate obtained from cows synchronized through one shot prostaglandin and inseminated with Tharparkar bull semen diluted with BIOXcell TM (supplemented with 0.02mm of vitamin E (α-tocopherol). Post-thaw motility of Tharparkar bull was observed in BIOXcell TM and Tris based egg yolk extender supplemented with and without vitamin E (α-tocopherol) 0.02 Mm/ml. The spermatozoa showed improved post-thaw results in BIOXcell TM supplemented with vitamin E (α-tocopherol) 0.02 Mm/ml, which ranged up to 58.31±0.86 and the results of motility of the current study agreed with the results obtained by Towhidi and Parks (2012), Kaka (2015) and Yadav et al. (2019). Their observed means were 61.67±0.59, 51.50±0.6 and 60.08±0.64. However, the value was lower than the findings observed by Motemani et al. (2017) 64.1±1.6. Meanwhile higher than the observations of Towhidi and Parks (2012), Ansari et al. (2012), and Kaka et al. (2015aKaka et al. ( , b, 2016, their recorded mean of findings was as 41. 4±0.4, 48.54±1.6, 45.5±0.2, 48.94±0.83 and 48±1.0. The fluctuation in results might be due to the use of antioxidants at various concentrations and processing techniques. However, Towhidi and Parks (2012); Kaka (2015), Kaka et al. (2015bKaka et al. ( , 2016 and Yadav et al. (2019) studied motility in BIOXcell TM extender at various concentrations of vitamin E (α-tocopherol).

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Post-thaw morphology of Tharparkar bull semen was also greater in the group of BIOXcell TM supplemented with vitamin E (α-tocopherol) 0.02 Mm/ml. Which obtained results ranging from 76.22±1.04. Current findings agreed with Ansari et al. (2012) and Motemani et al. (2017), 75±3.1 and 75±1.7. However, the value means the value of the present study is higher than the results carried out by Kaka (2015), and Kaka et al. (2015aKaka et al. ( , b, 2016. Their results were 71.00±1.7, 72±1.2, 70.63±0.54 and 66.25±3.4. The difference in results may be due to processing techniques, the use of extenders with different compositions, or it depends on the use of antioxidants. Meanwhile, the use of various concentrations of antioxidants presents variations in results. Meanwhile, Ansari et al. (2012) and Motemani et al. (2017) studied morphology in BIOXcell TM extender at various concentrations of vitamin E (α-tocopherol).
In the last membrane integrity was also highest in group BIOXcell TM supplemented with vitamin E (α-tocopherol) 0.02 mM/ml. The findings were 64.68±1.43. The result of the present study agreed with the results of Motemani et al. (2017), 61.7±1.6 and lower than Kaka (2015), and Kaka et al. (2015a, b), 70.00±2.9, 75±1.6 and 74.84±1.8, respectively. Meanwhile, the value was higher A. Channo et al.

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Semen Quality Parameters of Tharparkar Bull Semen Extended in BioXcell and Tris-Based Egg Yolk 5 than Ansari et al. (2012), 60.11±2.3. These variations in research findings may be due to variance of management, use of antioxidants at various concentrations and another handling/ processing factors (Sansone et al., 2000).

CONCLUSION
On the basis of the current study, it concluded that BIOXcell TM and Tris-based egg yolk extender supplement with vitamin E (α-tocopherol) showed improved postthawed quality parameters of Tharparkar bull semen.

ACKNOWLEDGMENT
The Author cordially acknowledges to Department of Animal Reproduction, Faculty of Animal Husbandry and Veterinary Sciences, Sindh Agriculture University, Tandojam, 70060 and Supervisor Dr Asmatullah Kaka (Assistant Professor, Department of Animal Reproduction, Faculty of Animal Husbandry and Veterinary Sciences, Sindh Agriculture University, Tandojam, 70060).

Funding
The research was carried out under the project "Optimization cryopreservation Protocol for Indigenous (Thari/ Tharparkar) Cattle Semen" funded by Sindh Agriculture Growth Project, Government of Sindh.

IRB approval
The work was approved by the departmental board of studies (BOS), Department of Animal Reproduction, Faculty of Animal Husbandry and Veterinary Sciences Sindh Agriculture University on 18th November 2021.

Ethics statement
All procedures in this study were conducted in accordance with approved ethical policies and protocols of SPU and Animal Reproduction Farm under Department of Animal Reproduction, Faculty of Animal Husbandry and Veterinary Sciences, Sindh Agriculture University Tanddojam.

Statement of conflict of interest
The authors have declared no conflict of interest.