Sperm quality and fertility of Murrah and Nili-Ravi bulls compared with indigenous buffalo bulls in Bangladesh

Abstract

Introduction

The livestock sector of Bangladesh is going through healthy positive steady growth over the last few decades with the farmer-friendly Government policy and robust and combined approach of farmers and entrepreneurs. Per-capita milk availability has increased in recent years and now 193.38ml milk/person/day is available against the FAO recommended quantity of 250 ml (DLS 2021). Although milk production has increased, still the position of Bangladesh is at the bottom of the global milk production list. On the contrary, the supply of milk is not evenly distributed among the populations as well as geographical locations. Furthermore, along with the increasing population rate of Bangladesh, milk demand increases by 4% per year against the annual milk production growth rate (3.6%) (Uddin et al. 2011). About 24.5 million cattle contribute 90% of total milk production, 3-4% comes from 1.5 million heads of buffalo and goat shares the rest (DLS 2013). Neighboring country India has had a steady growth in milk production over the last few years and about half (49.2%) of the entire milk comes from buffalo (Department of animal husbandry and dairying, Government of India, 2019). Although, Bangladesh is sufficient in meat production whereas only 0.1 million metric tons of meat is produced from buffalo in comparison with cattle (0.4 million metric tons) (Fatema, 2014). So, there is a huge opportunity to explore the milk and meat production potentialities of buffalo and to increase their contribution to the national economy. But the average milk production (600-1000/L, 250-270 days lactation period) potentialities of indigenous buffalo is the main challenge to achieving the targets (Huque et al. 2012). Research is going on to increase the productivity of indigenous buffalo. To support the research and development of buffalo production, the government is implementing different development projects on buffalo. However, Bangladesh has no specialized buffalo breed so far with increased milk and meat production potentialities. So, options for improving the productivity of local buffalo are either crossbreeding with improved buffalo breed or selection or breeding within the indigenous stock. In this context, elite buffalo breeding bulls from Murrah and Nili-Ravi breed has been imported from India for frozen semen production for crossbreeding with indigenous buffalo.

Good quality semen from pedigree-tested breeding bulls is the prerequisite of a successful crossbreeding program. This prerequisite is mostly hindered by the limitation of the availability of good quality breeding bull as well as quality semen. Frozen semen production has several inherent and functional constraints. Another challenge is the poor libido of buffalo bull and less quantity of semen production with the good number of viable spermatozoa. Furthermore, semen production is not constant throughout the years and smaller testicular size, less quantity of semen production and epididymal sperm reserve in buffalo bull are the natural constraints that make things worse than in cattle (Suryaprakasam and Narsimha, 1993; Sudheer and Xavier, 2000; Singh et al. 2003). Therefore, the expected frozen dose producing ability per bull is to be calculated, which will help in planning semen station establishment to meet the future growing demand for AI considering all the factors affecting semen production.

Semen analysis seems to be the most prolific diagnostic tool to evaluate male fertility potential, although there are other advanced tools available but it demands more technical knowledge and laboratory setup (Verstegen et al. 2002) Fertility is the most complex science and there are no specific set rules proved to be accurately reliable to predict fertility as both male and female and other environmental factors involve in successful fertilization. The conventional microscope-based semen evaluation is sometimes not precise and accurate (Patel et al. 2012), whereas computer assisted sperm analysis (CASA) is a quite faster and more precise tool to evaluate the concentration, motility, morphology and kinematic properties of sperm (Kumar et al., 2018).

Therefore, before mass production and AI at the farmer's level, semen quality and fertility potentialities of the imported bulls in comparison with indigenous bull needs to be evaluated. With this objective, the present study was designed to investigate the fresh and frozen semen quality and fertility potentialities of Murrah, Nili-Ravi and Indigenous buffalo bulls through CASA as well as to predict the quantity of frozen semen production potentialities of the bulls.

Materials and Methods

2.1 Buffalo bull Selection and management

Fourty-five (45) ejaculates from 6 breeding bulls (2 bull/group) of three groups of buffalo (Murrah, Nili Ravi, and Indigenous buffalo) were used for the experiment. The bulls (age 3-5 years) were individually housed in an intensive housing system at the Buffalo research farm of Bangladesh Livestock Research Institute (BLRI), Savar, Dhaka, Bangladesh and their overall management was uniform. Pure Murrah and Nili-Ravi bulls were imported from India and Indigenous buffalo bull was used from BLRI Buffalo breeding stock.  

2.2 Semen collection and evaluation

Buffalo bulls were trained for semen collection through an artificial vagina (AV) set before starting the experiment. Semen was collected once/week regularly in the early morning (6-6.30 AM) with an AV set (Walton, 1945). Collected semen from each bull was transferred to the laboratory for evaluation and further processing. Motility, morphology, concentration and kinematics were measured using a Computer Assisted Sperm Analyzer (CASA) (Hamilton Throne, IVOS II) (Miraz et al. 2022). The CASA settings for analyzing sperm motility were set as; Frame rate 60Hz, Frames acquired 30, Minimum contrast 35, Minimum cell size, 5 pixels, Cell size, 9 pixels, Cell intensity 110 pixels, Path velocity (VAP) 50 μm/s, Straightness (STR) 70%, VAP cut-off, 30 μ/s and VSL cut-off 15 μ/s. Prepared semen (1 μl) sample was loaded on a prewarmed Leja® slide and analyzed for sperm motility and kinematics. Besides total motility (%), progressive motility (%), static motility (%) and slow motility (%) of spermatozoa (Fig. 2) the following kinematic properties were also evaluated (Table 1 & Fig. 1): 

Table 1: Sperm kinematic parameters measured by the CASA system (Van der Horst, 2020) 

2.3 Semen dilution, equilibration and freezing

Fresh semen from three different genotypes of bulls were diluted with AndroMed (Minitube, Germany) diluter to prepare a concentration of 80x106 spermatozoa/ml. Diluted semen was equilibrated in a cold handling cabinet at 4°C for 4hr. After equilibration diluted semen was filled in a 0.25ml French mini-straw (20 million spermatozoa/straw) and sealed with an automated filling and sealing machine (MPP Uno, Minitube, Germany). Finally, semen was frozen with a programmable bio-freezer (Turbo Freezer M, Minitube, Germany) with liquid nitrogen vapor and frozen semen was plunged into liquid nitrogen (−196°C) can for storage. 

2.4 Post Thaw evaluation and artificial insemination (AI) 

Post-thaw evaluation of cryopreserved semen was conducted after 24 hours of storage. Frozen semen samples from each of the three genotypes were evaluated by CASA for motility, morphology and kinematic properties as described above as fresh semen. Artificial insemination (AI) was conducted with frozen semen in naturally estrous indigenous buffalo cows at their second and third parity. After 60 days post-AI, inseminated animals were checked for pregnancy with rectal palpation and the non-return rate was calculated. Artificial insemination and pregnancy diagnosis was conducted by the same technician to avoid the variation in conception rate. 

2.5 Statistical analysis:

Statistical analyses were performed using Statistical Package for the Social Sciences SPSS (25.0). One-way ANOVA followed by Duncan multiple range post-test was used to assess differences among the mean of motion characteristics of fresh and frozen semen among three groups and their sperm morphology, kinematics and dose/ejaculate. P value (p<0.05) was considered statistically significant.

Results

Volume and concentration

Freshly ejaculated semen from three groups of buffalo bulls was evaluated and quantified. There were no significant differences among the mean volume among the groups, although the Murrah bull (3.04±1.81) produced a higher quantity of semen followed by Nili-Ravi (2.67±0.44) and Indigenous bulls (2.26±0.40). The concentration of fresh semen was measured by CASA and no significant variations were observed among the group (p>0.05). The concentration pattern among the group follows the same pattern as volume (Fig. 3).

Sperm motility

The motility characteristics of fresh and frozen-thawed Murrah, Nili-Ravi and indigenous bulls were estimated by CASA and the results are presented in Table 2. In fresh semen, total, static and slow motility differed significantly among three different groups of bulls (p<0.01). Indigenous buffalo bull produced semen with higher total motility and lower static motility followed by Murrah and Nili-Ravi bull. However, no significant difference was observed for progressive motility among the bulls irrespective of group. After cryopreservation, total and static motility patterns remain the same with significant differences (p<0.01) among different groups of bull followed by Indigenous, Murrah and Nili-Ravi. However, after cryopreservation progressive motility differs significantly among the groups (p<0.01).

Table 2: Fresh and post-thaw sperm motility (mean±SD) of Murrah, Nili-Ravi and Indigenous bull

^{**=significant at 1% level, *= significant at 5% level, NS=Non- significant, Sig. = Significant}

^{Mean values in the same row with different superscripts (a, b, c) differ significantly (P<0.01 or p<0.05)}

Sperm morphology

Different morphological properties of fresh and frozen-thawed sperm was evaluated by CASA and presented in Table 3. In fresh semen, significantly higher bent tail % and distal droplet % were found in Nili-Ravi and Murrah bulls, whereas, semen from indigenous bulls has a lower bent tail and distal droplet percentage (p<0.01). Although there was no significant variation of Distal Mid-piece Reflex (DMR)% in fresh semen after freezing, indigenous bull's semen showed significantly higher DMR% (p<0.01).

Table 3: Fresh and post-thaw sperm morphology (mean±SD) of Murrah, Nili-Ravi and Indigenous bull

^{**=significant at 1% level, NS=Non- significant, Sig= Significant}

^{Mean values in the same row with different superscripts (a, b, c) differ significantly (P<0.01)}

Sperm kinematics

The kinematic characteristics of fresh and frozen-thawed Murrah, Nili-Ravi and indigenous bulls was estimated by Computer Assisted Sperm Analyzer (CASA) and the results are presented in Table 4. In fresh semen, there was significant differences were found for VAP, VSL, STR, LIN and BCF among the groups (p<0.05). Semen from indigenous bulls have higher VAP, VSL, STR, LIN and lower BCF followed by Nili-Ravi and Murrah (p<0.05). In frozen semen, significant differences were found for STR, LIN, ALH and WOB (p<0.01) among the groups.

Table 4: Mean (±SD) values of sperm kinematics of fresh and post-thaw sperm of Murrah, Nili-Ravi and Indigenous bull based on total motile sperm

^{**=significant at 1% level, *= significant at 5% level, NS=Non- significant, Sig= Significant}

^{Mean values in the same row with different superscripts (a, b, c) differ significantly (P<0.01 or p<0.05)}

Comparative semen production performances

The total number of frozen semen dose/ejaculate produced from each group of the bull was calculated. There were no significant differences was found among the groups, although more number of doses/ejaculates can be produced from Murrah bulls (316.53±131.65) followed by Nili-Ravi (297.33±95.79) and Indigenous bulls (294.64±152.14). The expected number of ejaculates that can be frozen/bull/year is assumed 52.14 (Once/week, 52.14 week/year) so, the expected frozen semen doses/per year/bull could be 16503.87, 15502.79 and 15362.53 for Murrah, Nili-Ravi and Indigenous bulls respectively. 

Fertility evaluation

Frozen thawed semen from three different groups of bulls were used for AI in indigenous buffalo cow (n=78, Murrah-27, Nili-Ravi-24 & Indigenous-27) at BLRI buffalo research farm and bull fertility was calculated based on 60 days non-return rate (Fig 4). There were no significant differences among the groups for fertility rate following AI (p>0.05).

Discussion

Production of viable zygote and subsequent live offspring is the ultimate goal of spermatozoa. Ejaculated spermatozoa have to negotiate different physical barriers and challenges in the female reproductive tract after ejaculation to reach the site of fertilization. During this entire journey sperm morphology, motility and functional integrity is the most important attributes to successfully pass the events of acrosome reaction, capacitation and hyperactivation. Sperm from the different breeds and even different bull from the same breed may show variations in their sperm quality and that may affect their fertilizing ability. In this experiment, the semen volume of the Murrah buffalo bulls are in the same range (2.56 ml) as reported by Ramajayan (2016). Bhave et al. (2020) evaluated semen production and semen quality of indigenous buffalo breeds under hot semiarid climatic conditions in India and found 4.48 ml volume and 1.31×109 million/ml concentration for Murrah buffalo semen. In this experiment, the volume of Murrah bull semen is lower and the concentration is higher than Bhave et al. (2020), this may be due to the lower number of bulls used in this experiment where Bhave et al. (2020) used 154 bulls. Adeel et al. (2009) evaluated the effects of diets containing fats on fresh and frozen semen of Nili-Ravi bull and found 2.71±0.32 ml semen volume and 1358 ±238×106 million/ml concentration for fresh semen in buffalo bull without supplementing dietary fat. The semen volume and concentration of the three groups of bulls in the current experiment are almost similar to the result of Hoque et al. (2018), who evaluated the effect of showering on semen quality of indigenous buffaloes of Bangladesh and found 2.04 ml volume and 1374.31million/ml concentration in fresh semen. Although the overall volume was lower the concentration is higher in this experiment for all three groups of bulls so, the total dose of straw production was not affected. 

Variations in post-thaw sperm motility and kinematic properties may come from the fresh semen quality of individual bull and the cryopreservation process. Fresh semen samples with higher total and progressive motility are more susceptible to cryo-damage (Muino et al. 2008).  In this experiment, indigenous bulls have significantly higher total and progressive motility than Murrah and Nili-Ravi bulls and subsequently, post-thaw semen motility is also higher in indigenous bull semen. In different buffalo breeds, total motility of fresh semen ranged from 71-75% (Rao et al. 1996). Some studies reported above 78% motility of fresh semen in Murrah and Surti buffaloes (Singh et al. 2013). Ravimurgan (2001) and Tiwari et al. (2011) reported post-thaw motility means (50.5% and 53.7%) in Murrah buffalo bulls. Bhave et al. (2020) evaluated semen production and semen quality of indigenous buffalo breeds and found total motility of 74.05% in fresh Murrah semen. In this experiment, semen motility is higher because the CASA system was used to evaluate the motility which is more precise and accurate than the conventional assessment system. The mean total motility of fresh and post-thaw Nili-Ravi bull semen was found 72.1 ± 1.0 and 56.4 ±3.7 respectively (Adeel et al. 2009). In this experiment, the total motility of Nili-Ravi bulls was higher in fresh semen but lower in frozen semen (Adeel et al. 2009). Total and progressive motility of fresh semen was found at 84.64% and 64.64 % respectively in indigenous buffaloes of Bangladesh (Houqe et al. 2018). When indigenous buffalo semen was frozen using an androMed extender, total and progressive motility of fresh and post-thawed buffalo semen was found at 92.9% and 52.3% (Nayan et al. 2020). Different experiments measured motility with different microscopes and software set up and there are variations among different evaluators also, so motility variations of the same genotypes under different experimental conditions are expected.    

In this experiment, bulls from three different groups show different morphological abnormalities whereas indigenous buffaloes have fewer abnormalities than Murrah and Nili-Ravi bulls. This may be due to adjustment to the environment, as Murrah and Nili-Ravi bulls were imported from India and there is a variation in temperature, humidity, and management which may result in higher morphological abnormalities in Murrah and Nili-Ravi semen. Sinha et al. (2021) estimated the climatic effects on different morphology and kinematics parameter of Murrah bulls and summarized that climatic stress has significant effects on the bent tail, coiled tail, distal droplet and distal mid-piece reflex percentage. Stress on animal results in higher reactive oxygen species (ROS) production which induce cellular damage through structural and functional changes and that subsequently may result in abnormalities in sperm (Valeanu et al. 2015). Anilkumar et al. (2017) found a 30.75% overall spermatozoal abnormality percentage in the Toda buffalo bulls. Normally, threshold values for sperm abnormalities in frozen semen should be within 15% whereas tail defects should not be more than 10% (Koonjaenak et al. 2007).   All of the three genotypes of this experiment have higher sperm abnormalities but their tail defects were within the range.  

Sperm motility is associated with sperm kinematics, in general, the higher the motility higher the VAP, VSL, VCL, STR and LIN. In this experiment, fresh semen motility of indigenous bull was higher than Murrah and Nili-Ravi bulls and subsequently VAP, VSL, STR and LIN are also significantly higher in the indigenous bull.  This result is also supported by an earlier study, where Muino et al. (2008) reported that a higher ratio of progressive sperm in an ejaculate is more susceptible to cryopreservation. In general, there was a reduction of motion characteristics for all three groups of semen during collection to post-thaw stages. The reduced value of sperm kinematics from collection to post thaw stage may be due to holding time, equilibration time and cryo-injury during cryopreservation (Lessard et al. 2000). Curvilinear velocity (VCL) is the most crucial kinematic property that changes during the process of cryopreservation (Rasul, 2000). Whereas, straightness (STR) and linearity (LIN) are considered to the important primary parameter to evaluate post-thaw motility of sperm (Anel et al. 2003). In the present study, there was no significant difference in post-thaw VCL among the groups, and significantly higher straight and linear post-thaw sperm was found for the indigenous bull. This results infrared that, frozen semen produced from all of the three groups are suitable for use in AI where frozen semen from the indigenous bull is better than Murrah and Nili-Ravi bulls in terms of kinematic properties.

Information on frozen semen production capacity from individual genotypes and individual bulls is important for designing the production capacity of the AI center for a smooth supply of frozen semen throughout the year. Zafar et al. (1988) reported yearly production to be 8,412 semen doses per bull in Nili-Ravi buffalo bulls and Roy (2006) produced 5,147.48 doses/year/bull in Murrah bulls. Ghodasara et al. (2016) estimated 4535.8 doses/year/bull for Jaffrabadi bull. In this experiment, the number of dose/ejaculates/bull and the number of dose/bull/year were higher as the total motility and concentration were higher for fresh semen. However, this production efficiency will largely vary on collection interval, seasonal variation, freezing efficiency and bull management.

Conception rates achieved in this study was within the normal expected range (30–60%) in water buffalo (Singh et al. 2016). In the Murrah bull, a 60.2% conception rate was found following AI when fresh semen was collected from the farmer's doorstep (Singh et al. 2013). The overall average pregnancy (%) in buffaloes was found 43.55±4.90 when AI was conducted in estrous synchronized buffalo (kumar et al. 2016). The non-return rate following AI using frozen-thawed semen from cloned buffalo bull was found 46-50% (Saini et al. 2020). Conception is the final destination of ejaculated sperm and ovulated oocytes that largely deepens on semen quality, insemination technique, proper timing of AI and other management and environmental (Dalton et al. 2010). In our study, all the buffaloes were inseminated by the same technician to minimize the variations. Sperm motility and sperm energy status is highly correlated (Quintero et al. 2004) as highly motile sperm can swim rapidly to the site of fertilization with a better chance of conception (Muino et al. 2008). Sperm having higher VCL and ALH shows higher zigzag motility and cannot reach the site of fertilization (Holt et al. 1997). However, total motility and progressive motility had a 12.9% contribution to the overall pregnancy in buffalo (Kumar et al. 2016). In this experiment, although there were no significant differences in fertility rate among the groups, the higher fertility rate (70.37±6.42) in the indigenous group may be due to significantly higher progressive motility and kinematics of frozen semen of indigenous bull.

Conclusion

All three groups of bulls produced fresh semen with good freezable quality whereas indigenous bulls produced semen with better qualities than Murrah and Nili-Ravi bulls. Irrespective of differences in fresh and frozen semen quality of the bulls they are suitable for AI with a considerable fertility rate at least in on-station conditions. In terms of quantity of frozen straw production/bull/year, more doses can be produced from Murrah bulls with once/week collection frequency followed by Nili-Ravi and Indigenous bulls. Murrah and Nili-Ravi bull can be used with similar efficiency along with indigenous bull for AI to increase the productivity of indigenous buffalo of Bangladesh. However, more research needs to carry out to evaluate the efficacy of AI and fertility rate at the on-farm condition for mass-scale frozen semen production.

Declarations

Acknowledgments

The author gratefully acknowledges Bangladesh Livestock Research Institute and Buffalo Research and Development Project for laboratory and funding support to conduct the study.

Conflict of Interest

None to declare

Authors' contributions

All the authors have contributed significantly to the planning, executing and drafting of the manuscript. All the authors have given their consent to submit the manuscript in the Journal of Tropical Animal Health and Production.

MFH Miraz and GK Deb were involved in the conceptualization, designing and execution of the experiment, writing - of the original draft, and project administration.

SMJ Hossain and S Akter contributed to the data analysis, writing and editing of the manuscript.

Funding

This work was funded by Buffalo Research and Development Project, Bangladesh Livestock Research Institute, Savar, Dhaka, Bangladesh.

Ethical approval

The study does not include any in vivo animal experimentation. The semen samples were collected from the animals following the standard method of semen collection using the artificial vagina method.

Data availability

The relevant data has been included in the manuscript.

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