Livestock sector is an integral part of the agricultural economy of Bangladesh. The need for animal proteins is rising day by day. Enhancing the reproductive capacity of cattle, particularly bull fertility, is one of the effective ways to meet this ever-growing demand of Bangladesh. It contributes 3.10% of total GDP and 16.52% of total agricultural sector (DLS 2021-2022). Total population size of cattle in Bangladesh is 24.7 million (DLS 2021-2022) but most of the cattle in Bangladesh are non-descriptive types, which do not belong to any specific breed. In Bangladesh, government, autonomous, NGOs and few private farms are still working for genetic improvement of our cattle through AI. Sahiwal is one of the major breeds of South Asia, which is originated in Montgomery district of Pakistan, and is distributed in certain herds of Punjab and Rajasthan in India. The fact that other nations (such as Kenya, Tanzania, Australia, the West Indies, and Bangladesh) imported Sahiwal animals shows how important this breed is either for crossbreeding with their native breeds or for adding zebu genes to crossbred animals to create synthetic strains. The pure Sahiwal breed is becoming less and less common due to careless crossbreeding with exotic breeds. It has also been acknowledged, though, that crossbred animals are not very good at adjusting to their surroundings (Joshi et al., 2001; Garcia et al., 2003; Rehman et al., 2006). Therefore, it is crucial to concentrate on the ongoing assessment, selection, and multiplication of superior Sahiwal cattle germplasm. A chemical medium called semen extender is used to preserve, extent, and protect sperm cells from shocks while they are being processed, stored, and transported in preparation for artificial insemination. A good extender should maintain the mediums pH and osmotic pressure while supplying energy for the sperm cells metabolic processes. Additionally, it shields sperm cells against cryoshocks when they are stored in liquid nitrogen at a very low temperature (-196°C) (Foote et al., 2010). Egg yolks have a protective effect because they contain lecithin, or a related phospholipid, which can be found alone or in combination with protein. In addition egg yolk extender, which is used to dilute semen for preservation has important and effective factors on successful storage of spermatozoa.   

The complicated process of cryopreserving semen is dependent on a number of variables, including individual variances, the relationship between cryoprotectants and the extender time, the amount of time needed for chilling and equilibration, and the rate at which freezing and thawing occurs (Whaley et al., 2021). The freezing rate is one of the key elements that affects how long the cells may survive the freezing process. There is an ideal freezing rate for each type of cell that will allow it to survive the freezing process. According to Watson, cells that undergo delayed freezing to dehydrate are more likely to survive than those that undergo quick freezing, which increases the risk of cell death. The freezing rate during the cryopreservation of semen influences the spermatozoas post-thaw quality (Khan et al., 2021). Several workers studied different freezing rates to improve the quality of frozen semen of different animals (Nur et al., 2011; Sarma et al., 2015; Dalal et al., 2018). Although a number of studies have been conducted to assess Sahiwal semen, not much data is currently accessible. Therefore, the present study was designed with the objective to evaluate the frozen semen quality of Sahiwal breeding bull using two different types of diluters and freezing protocols. 

Experimental animals

In this study, semen samples were collected from Sahiwal Bulls (SL-75, SL-901, SL-381708). At American Dairy Limited (ADL), all of these bulls were kept in similar food and management circumstances (all bulls were fed with a standard daily diet consisting of 6 kg 16% protein concentrate pellets and 20 kg green fodder (Puckchong, Jerman & Maize; loose housing system 15×70 ft.; Ambient temperatures were varied from a minimum of 22°C to a maximum of 36.2°C during the experimental period. The average humidity in the area ranges from 66.50% to 80.68%). For this study, three breeding bulls were chosen as test subjects based on factors such as age, body weight, and scrotal circumference (Table 1). Semen was collected from those bulls once in a week (Friday).

Semen collection

In general, semen was routinely collected in the early morning. A regulated sexual preparation process preceded semen collection. Once the bulls arrived at the collection floor, they were placed in a waiting area where they could watch the other bulls being collected. They were permitted two false mounts spaced two minutes apart after their libido was aroused. Using a teaser animal-a bull renowned for his docility and robustness-the ejaculate was retrieved at the third mount using an artificial vagina (37°C). On the day of collection, one or two ejaculates were collected from each bull. 

Table 1: Description of the breeding bulls.

Fresh semen evaluation

By observing the collection vials graduated mark in milliliters, the ejaculate volume was measured. The concentration of spermatozoa was measured with a Density Spectrophotometer (SDM-6, Minitube, Germany). A 0.9% sodium chloride solution was used to dilute fresh semen in cuvettes at a ratio of 1:100. The reading was recorded from the Density Spectrophotometer in million/mL. The percentage of sperm motility was determined using the Computer Assisted Sperm Analyzer (CASA) Androvision minitube (Minitube, Tiefenbach, Germany). This encompassed both normal and abnormal movements. 

Evaluation of fresh semen 

The highest (11.13mL) volume of semen was obtained in SL-901 and lowest (10.26mL) in SL-75. The ejaculate volume of semen found in this investigation is consistent with the findings of Hafez (1974) and Hossain et al. (2022).  Kumari et al. (2019) and Hasan et al. (2020) reported 4.75±0.14 and 5.26±0.17mL of semen produced by Sahiwal bulls which is far less than from the present study. The age, maturity, nutritional state, overall health, endocrine balance, and soundness of the breeding bulls sex organs are among the other characteristics that significantly influence the variation in semen production (Peters, 1987).  Higher value of concen-tration was found 2364.90x106/mL in SL-075 which was significantly higher than bull SL-381708 (1966.23x106/mL) where SL-901 (2236.5723x106/ mL) stood an intermediate position. The present study strongly supports with the findings of Hasan who found the sperm concentration 2036.00±43.99× 106/mL in Sahiwal bull (Hasan et al., 2020). Kumari reported 1630.4±0.02×106/mL, 1471±37x106/mL and 1354±19.34x106/mL in Sahiwal which are significantly less than those of the current study (Kumari et al., 2019). The differences in age, feeding schedule, sperm collection frequency, climate, semen collector competence, and AV temperature could all be contributing factors to the observed sperm concen-tration discrepancy.  Motility percentage 92.70 in bull SL-901 was significantly higher than that of SL-075 and SL-381708. Ansari reported 69.6% motility for fresh semen of Sahiwal which are comparatively lower than the present study (Ansari et al., 2010). Sperm motility is an accurate indicator of sperm viability and fertility (Janett et al., 2008).  The research data stated that the percentage of progressive motility of semen was significantly higher in the three Sahiwal breeding bulls which is an indication of good quality semen. Khan observed the progressive motility in fresh semen in Sahiwal bulls (20.25± 0.47%) which is far less than the present study (Khan et al., 2018). The rapid progressive motility was observed in SL-075 (83.09%) significantly (P<0.01) higher than the bull SL-381708 (55.30%). In general, it is thought that the most reliable indicator of sperm motion for estimating a semen samples ability to fertilize is rapid progressive sperm motility. Rapid progressive motility is necessary for spermatozoa to pass through cervical mucous efficiently (Bjorndahl, 2010).

The mean VSL of SL-075 (63.32µm/s) was significantly (P<0.01) higher than SL-901 (52.16µm/s) and SL-381708 (50.75µm/s). The highest value 152.34µm/s of SL-075 was reached in significant level (P<0.01). The most crucial kinematic characters to assess are VCL, VAP, and VSL, which are positively correlated with spermatozoas capacity to fertilize (Jobling et al., 2002). Improved conception rates in farm animals are indicated by spermatozoa exhibiting higher values for these characteristics. The mean LIN of SL-381708 (0.45µm/s) was significantly (P<0.01) higher than the other bull. Greater linearity % results indicate that spermatozoa have a greater ability to migrate through the female reproductive canal and that they are transported to the site of fertilization on time (Cox et al., 2006).  Sperm morphology was observed in 93.87, 93.87 and 93.60% in bull SL-75, SL-901 and SL-381708, respectively. Normal and live sperm percentage in fresh semen sample represents good quality semen. The observation is lined with Herman who showed that good quality sperm must have 80 percent live sperm. The findings of the current study are found slightly higher than the result of Akhter who recorded the live spermatozoa percentage of Sahiwal cross was 82%. This variation may be due to missing of normal spermatozoa and error during slide preparation (Akhter et al., 2013). Morphological abnormality was recorded 6.13, 6.27, 6.33% of bull SL-75, SL-901, SL-381708. It reveals that the sperm quality is good and it can be used for frozen semen production. The present result is highly supported by Roberts opinion that the total sperm abnormalities in a viable semen sample should not exceed 20 percent. Mondal et al. (2005) found the total abnormal sperm (18.40±3.03%) in Sahiwal that is higher than the present study. The sperm abnormalities may differ depending on the methodology used, temperature shock, and collection method.

Frozen semen evaluation

Effect of diluter on frozen semen quality 

Tris citric acid, D-fructose, Antibiotic (Cocktail), Glycerin and Egg-yolk used in one step diluter for preparing frozen semen which preserve the fertility of animal sperm at high extension rates. The highest post thawing total motility, progressive motility, progressive fast motility, circular motility, curvilinear velocity (VCL), straight line velocity (VSL) are obtained 78.42, 73.50, 59.68, 0.77, 115.31, and 44.83%,  respectively using diluter 1 and the lowest obtained using diluter 2. Some other parameters like slow motility, local motility, linearity were found higher using diluter 2 than diluter 1. These findings shows that diluter 1 possess better result than diluter 2. Sperm motility is one measure thats frequently used to evaluate the quality of semen. Some parameters of sperm kinematics relate to fertility (Oliveira et al., 2013) including curvilinear velocity (VCL), straight line velocity (VSL). The VCL and VSL values needed for sperm to be able to penetrate the ovum are VCL >70 µm/s and VSL >45 µm/s (Inanc et al., 2018). One-step dilution yields higher results than two-step dilution for all the parameters that are commonly used to evaluate semen quality and are related to fertility, such as post-thawing total motility, progressive motility, progressive fast motility, curvilinear velocity (VCL), and straight line velocity (VSL). This observation lined with Arif who showed one-step dilution gives better result than two and three step dilution and contradicts with (Arif et al., 2020). Berghe, who demonstrated a considerable improvement in sperm cryopreservation using a two-step dilution tris-egg yolk extender containing Equex STM (Berghe et al., 2018). The reasons of being difference with the present findings may be due to diluter composition, freezing protocol and species variation. Major fluctuations in osmotic pressure occur in sperm during freezing and thawing. As they interact with the freezing and thawing speeds, the basic extender utilized and the cryoprotectant concentration determine how stressed the sperm membranes are.

Effect of freezing protocols of frozen semen quality 

In the current study, a significant difference was found for some parameters like slow motility, curvilinear velocity (VCL) and linearity. This fluctuation could be caused by variations in the freezing process, diluter, and raw semen quality. Other parameters studied in this findings like post-thawing total motility, progressive motility, progressive fast motility, circular motility and straight-line velocity, there is no significant difference found between two different freezing protocols. In case of freezing protocol 1 (5 steps), this observation is lined with Ennen and Gilbert demonstrated the advantageous benefits of gradually cooling diluted semen to 5°C before freezing, produced comparatively better results. In case of freezing protocol 2 (2 steps), the results of the present study collaborate with Galarza who reported that two-step accelerating cooling rate yielded lowest cryoinjury to ram sperm cells (Galarza et al., 2019). This discovery aligns with the findings of Barbas et al. (2009) and Brito et al. (2017). This observation, however, is not in line with that of Hassan et al. (2019) who demonstrated that slow freezing produces results that are generally superior to those of fast freezing. According to Pugliesi et al. (2014), bovine spermatozoa in straws can withstand a broad range of freezing rates without significantly affecting their motility after thawing.

In this study, freezing protocol-1 (Minitub Turbo TM) works perfectly due to quardiple nitrogen injection, static and forced vapor with quardiple fan distribute uniform nitrogen vapor very accurately in biofreezer chamber when freezing. For these reason, freezing protocol-1 gave good result and very close to each other. On the other hand, freezing protocol-2 (Imv Digicool-5300) is not perform satisfactorily due to mono injection of nitrogen with single fan along with bottom via top vapor system which was responsible for lower motility and velocity parameter also.

Correlation among semen parameter

The volume and concentration showed antagonistic correlations of -0.17 for SL-075; this is consistent with the findings of (Gredler et al., 2007), who used 301 Austrian dual-purpose Simmental (Fleckvieh) AI bulls to achieve a correlation value of -0.17 between volume and sperm concentration. There was also a positive correlations found between semen volume and concentration which was 0.07 and 0.36 for SL-901 and SL-381708 which agrees with Mia et al. (2013). A negative correlation between semen volume and total motility was -0.87 and -0.02 for SL-075 and SL-381708 which lined with Druet and a positive relationship was found for SL-901. Semen volume, progressive motility, VSL has a positive correlation in bull SL-075 and SL-381708 and negatively correlated in case of bull SL-901 whereas semen volume is negatively correlated with VCL in bull SL-075 and SL-381708.The correlation between concentration and motility was 0.34 and 0.62 for SL-075 and SL-381708 which agrees with Druet et al. (2009). Druet also showed that concentration and progressive motility has a positive relation which agrees in case of bull SL-381708 but contradicts for bull SL-075 and SL-901. Concentration has positive relationship with VCL which agrees with Ertas who showed a positive correlation between sperm velocity and its concentration which contradicts negative correlation between concentration and VSL (Ertas et al., 2019).

In the present study, there was a positive and negative correlation found between sperm motility and velocity. The findings of this study collaborate with Farrell. According to Hirano et al. (2003), there was a significant correlation between the sperm velocity measured by SQA V and several of the CASA estimates, such as sperm motility (p = 0.001), the percentage of progressively motile sperm (p<0.001), straight-line velocity (P<0.001), curvilinear velocity (P<0.001), and average path velocity (P<0.001). A strong positive correlation was found between VSL and VCL which was 0.54 for SL-901 and 0.15 for SL-075. The positive correlation between the velocity parameters and other CASA variables indicates that spermatozoa have a higher potential for fertilization (Cremades et al., 2005). Consequently, semen samples containing these spermatozoa have higher rates of pregnancy and fertility following artificial insemination. The breed, nutritional status, management techniques, and variations in climate might all be blamed for the variation.

The frozen semen quality using diluter 1(tris egg-yolk) gives comparatively better result than diluter 2 (tris egg-yolk aqua). On the other hand, there is no significance difference found in case of evaluation of maximum parameter using freezing protocol 1 (slow freezing) and freezing protocol 2 (fast freezing) so both may be used for frozen semen production. From this study it might be recommend to use diluter 1, freezing protocol 1 and freezing protocol 2 to get better result for frozen semen production of Sahiwal bull. Among semen characteristics, the strongest positive associations were seen between overall motility and sperm concentration and VCL (0.62 and 0.67) and semen volume and VSL were (0.51) for SL-381708 means that spermatozoa have higher rate of fertilization potentiality. According to the evaluations findings, Sahiwal bull semen appears to have a lot of potential for producing frozen semen.

This research complies with the ethical standard required for the research concerning the handling of biological material. The approval number: Ref: 1711/BAURES/ESRC/39/2024. 

S.S.H.; and M.A.M.Y.K.: conceived and designed the experiment. N.T.: performed the study and conducted lab analyses. M.R.I.; and S.S.H.: supervised and coordinated the experiments and provided clinical data. M.R.I.; and M.M.A.: performed statistical analyses of experimental data. M.M.A.: prepared the draft of the manuscript. All authors critically revised the manuscript and approved the final version. 

I am very thankful to the department of Animal Breeding and Genetics, Bangladesh Agricultural University, Mymensingh for logistic support and Government of Peoples Republic of Bangladesh for providing financial support.

There is no conflict of interest to declare.