Frozen semen quality and fertility of imported pure Holstein Friesian and Sahiwal breeding bulls in Bangladesh

The present study was conducted to evaluate the frozen semen quality in terms of different motility patterns and fertility of imported pure Holstein Friesian and Sahiwal breeding bulls maintained under farming condition of Bangladesh. 100% pure Holstein Friesian and Sahiwal breeding bulls were imported and thereafter reared in the American dairy limited (ADL). Semen samples were collected once a week, processed and frozen in the laboratory of ADL. On the day of collection, semen volume and sperm concentration measured immediately after collection. Different motility patterns of spermatozoa (progressive, fast, slow, local, immotile) were measured by Computer Assisted Semen Analyzer (CASA). Effect of breed was found significant (p<0.05) on semen volume and sperm concentration. The semen volume (ml/ejaculate) and sperm concentration (×10) were found to be 6.13±0.28 and 1664.28±62.41 in Holstein Friesian breeding bulls whereas 5.26±0.17 and 2036.00±43.99 in Sahiwal bulls, respectively. The progressive motility, fast motility, slow motility and local motility were significantly (p<0.05) higher in Holstein Friesian bulls than that of Sahiwal bulls both before and after freezing. Different motility patterns such as progressive, fast, slow, local and immotile motility were found to be 89.22±0.45%, 75.97±0.67%, 9.10±0.36%, 4.25±0.15% and 10.05±0.41% in Holstein Friesian bulls whereas 84.98±0.89%, 65.99±0.92%, 11.88±0.37%, 5.65±0.19% and 15.69±0.54% in Sahiwal breeding bulls, respectively before freezing. On the other hand, after freezing, the progressive, fast, slow, local and immotile spermatozoa were found to be 68.19±0.46%, 53.39±2.01%, 9.21±0.46%, 8.47±0.72% and 26.76±2.01% in Holstein Friesian bulls whereas 56.54±0.25%, 42.52±0.67%, 11.11±0.55%, 13.04±1.24% and 30.45±1.58% in Sahiwal bulls, respectively. Non-return rate was found insignificant between the breeds and it was 64.19% in Holstein Friesian and 64.71% in Sahiwal bulls. In conclusion, frozen semen quality of Holstein Friesian pure breed is better than that of Sahiwal breeding bulls but fertility of both breeds are similar. Therefore, frozen semen of both breeds can be used in the field level for the genetic improvement of cattle.


Introduction
Cattle population in Bangladesh encompasses mostly non-descript types with a few varieties namely Red Chittagong, Pabna, Munshiganj, and North Bengal grey cattle (Hossain et al., 2006). They are smaller in size and their milk production capacity is much lower. The basic aim of cattle breeding program in Bangladesh is to improve the genetic potentiality of local cattle through infusion of exotic gene (Ahmed and Islam, 1987). There are two mating system practiced in our country such as natural and artificial. Having multiple benefits of Artificial Insemination (AI) over natural mating, it is getting popularity in our country. AI is the most economical and management tool as it provides the opportunity to spread the superior germplasm by the extensive use of progeny tested selected sires. On an average, a bull can produce fifty thousand progeny per year through AI compared to 40-50 progeny through natural mating (Salisbury et al., 1978). Hafez (1993) pointed out that Artificial insemination is the most important single technique ever devised for the rapid genetic improvement of animals. AI plays an important role to increase the yielding capacity of cows and is the appropriate and cheapest way of genetic improvement of cows. Male selection is very much crucial in case of breeding. The biological and economic importance of a bull's contribution through natural breeding or artificial insemination (AI) for improving reproductive efficiency and production of meat or milk, or both is of great importance because each bull or its semen represents half of the genetic composition of its progeny (Foote, 1979). Male can serve many females with single ejaculation using artificial insemination and produce many offspring. Bull selection must be accurate otherwise the whole system will be collapsed. Any wrong decisions in bull selection can haphazard the whole system. For the economic viability, caution about poor quality bulls in terms semen quality should be taken into consideration. If there is problem in fertility due to fault of bulls, it will cause huge economic loss to the farmer. When only highly fertile bulls are used, better fertility are achieved, which reduces the costs of reproductive programs (Sudano et al., 2011). Evaluation of semen parameter is a good indicator for judging the bull's reproductive performance. The term 'quality of semen' means package of parameters that represent the inner picture of semen related to fertility. Different factors including the type of extender, interactions between cryoprotectants, cooling rate, thawing rate and the physiological status of animals are important for successful semen cryopreservation and AI. Moreover, preservation process can also affect the quality and fertility. Semen preservation might be for either short tome (chilled) or long time (frozen). Frozen semen is replacing chilled semen and used all over the world due to some limitations of using chilled semen at the remote areas and for long time. On the other hand, in case of frozen semen, semen quality may deteriorate after freezing. So, it is necessary to ensure the semen quality in terms of different semen parameter after freezing. In Bangladesh, a substantial number of crossbreds have been produced so far using the exotic gene pool of 50% blood level of Holstein-Friesian, Sahiwal, Sindhi, Jersey breeding bulls. However, to make a white revolution in the country and meet up the huge demand of milk and milk products, pure Holstein Friesian and Sahiwal breeding bulls have been imported in Bangladesh by different private organizations and entrepreneurs. Their aim is to disseminate the genetic merit of these pure breeding bulls though frozen semen and genetic improvement of our cattle. But no research works have been performed before to evaluate these breeding bulls and their frozen quality and fertility in the context of Bangladesh. From this point of view, our aim is to evaluate the frozen semen quality and fertility of these imported pure Holstein Friesian and Sahiwal breeding bulls maintained in the farming condition of Bangladesh.

Selection of breeding bulls
100% Holstein Friesian (HF) and Sahiwal (SW) pure breeding bulls have been imported and maintained in American dairy Limited, Vagnahati, Sreepur, Gazipur. Among them, three Holstein Friesian and three Sahiwal breeding bulls were selected on the basis of age, body weight and scrotal circumference (SC) and used for this research. The ages of these breeding bulls ranged from 4 to 6 years, body weight from 1048 to 1134 kg and scrotal circumference from 34 to 37 cm for HF bulls. In case of SW bulls, age ranged from 4 to 5 years, body weight 600 to 645 kg and SC was 34.5 to 37.0 cm, respectively. Bulls were maintained in intensive farming condition and supplied with balanced ration comprising concentrate and roughage depending on their body weight. Fresh water was made available all the time.

Preparation of diluter
Egg yolk-citrate (EYC) diluter was prepared at previous night of collection of semen. It was a solution of 2.94% sodium citrate in 100 ml of distilled water and in which 100000 IU of penicillin and 100000 µg of streptomycin were added. One parts of egg by volume was added with three parts of the sodium citrate solution. It was stored in proper temperature and used after overnight preservation.

Semen collection and evaluation of fresh and diluted semen
Semen was collected by means of artificial vagina (AV) method once in a week. Ejaculate volume was measured using graduated collection vial tube in milliliter. The concentration of spermatozoa was determined by Density Spectrophotometer (SDM-6, Minitübe, Germany). Fresh semen was diluted in cuvettes with 0.9% sodium chloride solution at the ratio of 1:100. The reading was recorded from the Density Spectrophotometer in million/ml. After that fresh semen was diluted with prepared EYC diluter. Progressive motility, fast motility, slow motility, local motility and immotility of diluted semen were measured by using Computer Assisted Semen Analysis (CASA) system (Andro Vision AX10, Minitübe, Germany).

Processing for frozen semen production
Diluted semen was filled with automatic filling, sealing and printing machine (MPP Quattro, Minitube, Germany) that could fill 4 straws at a time. For equilibration, semen straws were decorated in the racks and placed in the cold handling cabinets named cooling castle maintained at 4-5º c for 3-4 hours. Semen straw was transferred from cold handling cabinets to Bio freezer (TurboFreezer L, Minitube, Germany). After keeping in the chamber of device, temperature was controlled by programmable software. Temperature curves and data of each freezing cycle could be stored, displayed and transferred into excel for further analysis. Subsequent freezing cycles started immediately after the previous freezing. The unidirectional nitrogen gas flow in the freezer ensured that all straws passed through identical freezing curves. After freezing one (1) or two (2) straw were taken per batch and analyzed to know the status of progressive, fast, slow, local motility and immotility of the spermatozoa by using CASA system and then transferred to large cryocan maintained at -196º c for preservation.

Artificial insemination and fertility measurement
Artificial insemination (AI) was performed in different areas of Mymensingh districts using the frozen-thawed semen of pure Holstein Friesian and Sahiwal breeding bulls. Bull fertility was measured in terms of Non-return rate (NRR). NRR was recorded by the percentage of cows that were not returned to estrus at 60 days after AI and calculated in percentage using the following formula.

Statistical analysis
The data obtained from the experiment were entered in Microsoft Excel worksheet, organized and processed for further analysis. Analysis was performed with the help of Statistical Analysis System (SAS, 2009) computer package.

Evaluation of fresh semen
Means along with standard error (SE) of volume and sperm concentration of the experimental breeding bulls are shown in Table 1. Semen volume of Holstein Friesian (6.13±0.28 ml) was significantly (p<0.05) higher than that of Sahiwal breeding bull (5.26±0.17 ml). The number of spermatozoa per ml semen of Sahiwal bull (2036.53±43.99×10 6 ) was significantly (p<0.05) higher than that of Holstein Friesian (1664.28±62.41×10 6 ) bull.

Comparison of pre-and post-freezing motility pattern of spermatozoa 3.4.1. Progressive motility
The progressive motility ranged from 88-89% in pre-freezing diluted semen which decreased significantly (p<0.05) in the post-freezing evaluated semen (60 to71%) in different pure Holstein Friesian breeding bulls (Figure 1). Similarly, freezing had a significant effect (p<0.05) on the progressive motility of Pure Sahiwal bull semen. Before freezing, the progressive motility of Sahiwal bull's semen ranged from 83 to 87% whereas after freezing, this percentage ranged from to 54 to 58% (Figure 1). Figure 2 showed that the fast motility of different pure Holstein Friesian breeding bull's semen ranged from 75-77% before freezing and it declined significantly (p<0.05) with a range of 42-68% after freezing. Similar trend was also observed in the fast motility of Sahiwal bull semen where pre-freezing fast motility of different Sahiwal bull semen ranged from 62-69% and post-freezing 40-45% (Figure 2).

Fertility of breeding bulls
Fertility of breeding bulls was expressed based on the cows not returned to estrus within the next 60 days after insemination. The average non-return rate did not vary significantly between these two breeds and it was 64.19% in Holstein Friesian and 64.71% in Sahiwal bulls ( Figure 3).

Evaluation of fresh semen
Considering the genotypic effect, it was found that the volume of semen varied significantly among the genotypes studied in this study. This observation strongly supports with the findings of the other published works (Brito et al., 2002;Ha et al., 2012). Islam et al. (2018) and Gopinathan et al. (2018) reported 5.63±0.16 and 4.53±0.05 ml semen per ejaculation in case of Holstein Friesian which is slightly lower than present study. Baloch et al. (2019) recorded 7.10 ml semen volume in Holstein Friesian which is slightly higher than this study. On the other hand, present findings on the semen volume of Sahiwal breeding bulls strongly support the previous research works ( Mostari et al., 2004;Latif et al., 2009). The variation in the ejaculate volume might be attributed to the genetic potentiality of the breeding bulls; scrotal circumference, body size and body weight the secretary activities of the sex glands (Leon et al., 1991;Hafez, 1993). Besides these, semen production also depends on several other factors such as the age, maturity, nutritional status, general health condition, endocrine balance and soundness of the sex organs (Peters, 2002). The concentration of spermatozoa per ml semen differed significantly (p<0.01) between the breeds of breeding bulls. The findings of the present study corroborate with results of other researchers (Hafez, 1983;Hossain et al., 2012;Rahman et al., 2014). According to Debnath (1999), the sperm concentration were 1354±19.34 million/ml in Sahiwal and 1204±19.34 million/ml in Holstein Friesian breeding bulls that are noticeably lower than the present study. According to Ha et al. (2012), the sperm concentration fluctuated between 1.06 to 1.34 billion/ml in Holstein Friesian. Ahmed et al. (2014) reported 1528.2±114.9 × 10 6 /ml and 1043.5±93.2 × 10 6 /ml sperm concentration in Sahiwal and Holstein Friesian breeding bulls. Such variations observed by various workers may be due to difference in age (Ahmad et al., 2003), environment, season (Sardar, 2007), breed, scrotal size, libido, sexual rest, frequency of ejaculation (Kumar, 1979), management, nutrition, physiological status of bulls and genetics (Mathevon et al., 1998).

Evaluation of diluted semen
Motility patterns of diluted semen are very much important to check the semen quality. The research observation is lined with Hoflack et al. (2006) and Galmessa et al. (2014). Islam et al. (2018) found 74.73±0.76% progressive motility in Holstein Friesian crossbred breeding bulls which corroborates with the present findings. However, present results are found slightly higher than the results of Morrell et al. (2018);Sundararaman et al. (2012); Rahman et al. (2014) and Hossain et al. (2012) Islam (2015). The reasons of being difference might be due to breed composition, diluter, and evaluation method.

Evaluation of frozen semen
The progressive motility of frozen Sahiwal bull semen reported in this study agrees with the findings of Lecewicz et al. (2015); Dolezalova (2016); Baloch et al. (2019) and Mostari et al. (2004). However, the findings of the present study are lower than the findings of some previous research works (Sugulle et al., 2006;Ansari et al., 2010;Gopinthan et al., 2018) and higher than the findings of Ulfina et al. (2002). The progressive motility of Holstein Friesian bull in this study is similar with that of Murphy et al. (2018) and Dias et al. (2018). Conversely, this progressive motility of Friesian bull reported in this study is less than that of Córdova-Izquierdo et al., 2015 andNasir et al., 2019. This difference might be due to raw semen quality, variation in diluter and freezing method.

Comparison of pre-and post-freezing motility of spermatozoa
Post freezing semen quality in respect of different motility patterns significantly reduced in both Holstein Friesian and Sahiwal breeding bulls. These pre-and post-freezing motility changes are in close agreement with Sonar et al. (2016) in Gir bulls; Pathak (2008) in Sahiwal and Red Sindhi bulls and Jersey × Red Sindhi bulls (Thakur et al., 2006). The post freezing motility value in the present study is lower than Jersey bulls (Thakur et al., 2006) while higher as compared to Friesian bulls (Mandal and Tyagi, 2007) and HF × Jersey × Kankrej bulls (Raval et al., 2007). Bhupal et al. (1993) reported that in identical environment, HF bull semen had better post freezing motility than that of Sahiwal bulls. Post-freezing motility can be affected by dilutors (Belorkar et al., 1993;Pramanik and Raina, 1998), method used for glycerol addition (Gilbert and Almquist, 1978;Arancibia et al., 1987) and equilibration time (Belorkar et al., 1993). All these factors might have contribution to the difference in observations by other workers for post thaw motility.

Fertility of breeding bulls
The findings of the present study is lined with Islam et al. (2018) who reported 63.67±1.46% non-return rate in Holstein Friesian crossbred breeding bulls. Moreover, the present findings coincide with the findings of other researchers (Nasrin et al., 2008;Rahman et al., 2014). Mostari et al. (2004) found 70.90±0.35% that is slightly higher than this study. Difference in fertility might be due to genetic makeup of cows and bulls, semen processing, thawing of straw prior to insemination, number of inseminations, quality of semen, site of semen deposition, proper timing of insemination and reproductive health of the female.

Conclusions
This study reveals that frozen semen quality in terms of different motility patterns of Holstein Friesian is better than that of Sahiwal breeding bulls but fertility of both breeds are similar in field condition. Therefore, frozen semen of both breeds can be used in field level for the genetic improvement of cattle. In addition, these results have given an insight view to the farmers, researchers, policy makers and entrepreneurs about the frozen semen quality and fertility of imported pure Holstein Frisian and Sahiwal breeding bulls at field condition in Bangladesh.