Journal of Animal Health and Production

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JAHP_7_4_171-178

 

 

Research Article

 

Effect of Honey as an Additive for Cryopreservation on Bull Semen Quality from Different Cattle Breeds under Tropical Condition

 

Eric Lim Teik Chung1,2, Nazri Nayan1, Nurul Syazana Mohammad Nasir1, Phen Syom A Hing1, Suriati Ramli3, Mohd Hafiz Abdul Rahman3, Mamat Hamidi Kamalludin1,2*

1Department of Animal Science, Faculty of Agriculture, Universiti Putra Malaysia, 43400 Serdang, Selangor, Malaysia; 2Institute of Tropical Agriculture and Food Security, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor, Malaysia; 3Institut Biodiversity Veterinar Kebangsaan, Bukit Dinding, 27000 Jerantut, Pahang.

 

Abstract | Previous studies have demonstrated that honey is a good additive for frozen semen, but the composition of honey is ultimately influenced by the plant species visited by the honey bees, the environment, processing, and storage conditions. Therefore, the main objective of this study was to determine the effect of using local honey derived from the Apis mellifera bee as an additive in Bioxcell™ extender for cryopreservation on bull semen quality from different cattle breeds. Different concentrations of honey additive in the commercial extenders (1, 2.5, 5, 10 and 15%) were prepared accordingly. Semen samples were collected from 12 sexually mature bulls consisted of three Mafriwal, three Jersey, three Peidmontese, and three Limousin bulls with the aid of an artificial vagina (AV) and a teaser cow. The semen samples were extended 1:1 (semen: extender) in the prepared extenders for cryopreservation. Semen evaluations were carried out on fresh semen, post-chilled, and 14th days post-thawed frozen semen. The semen samples were evaluated based on microscopic characteristics such as general motility, progressive motility, and liveability. The results showed that there were significant differences (P˂0.05) in the sperm quality between different breeds of bull and between different concentrations of honey additive extenders. Jersey bull exhibited the best sperm quality with the highest sperm general motility, progressive motility, and liveability throughout the fresh semen, post-chilled, and post-thawed frozen semen followed by Mafriwal, Piedmontese, and Limousin. The sperm quality of all bull species increased significantly (P˂0.05) by the 1% concentration of honey additive extender compared to 2.5, 5, 10 and 15% concentration of honey additive extenders. In summary, 1% concentration of honey additive extender can be used effectively in bull semen cryopreservation to preserve the sperm quality.

 

Keywords | Bull, Cryopreservation, Semen quality, Apis spp., Honey.

 

Received | June 20, 2019; Accepted | September 02, 2019; Published | December 28, 2019

*Correspondence | Mamat Hamidi Kamalludin, Department of Animal Science, Faculty of Agriculture, Universiti Putra Malaysia, 43400 Serdang, Selangor, Malaysia; Email: mamath@upm.edu.my

Citation | Chung ELT, Nayan N, Nasir NSM, Hing PSA, Ramli S, Rahman MHA, Kamalludin MH (2019). Effect of honey as an additive for cryopreservation on bull semen quality from different cattle breeds under tropical condition. J. Anim. Health Prod. 7(4): 171-178.

DOI | http://dx.doi.org/10.17582/journal.jahp/2019/7.4.171.178

ISSN | 2308-2801

Copyright © 2019 Kamalludin et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

 

Introduction

 

The success of a livestock industry depends on the genetic improvement and the number of production animal. Although the traditional breeding method is still being practiced successfully, reproductive biotechnology such as embryo transfer (ET) and artificial insemination (AI) have been introduced for genetic improvement and a better production (Aires, 2003). In cattle breeding, AI is the most widely used technique where bull semen is used to artificially inseminate a cow during heat. However, the fertility and performance of spermatozoa in fresh semen can only be maintained for a few hours. As a result, frozen semen is produced to preserve the spermatozoa from a good genetic animal for years without any changes in their metabolism and structure under ultralow temperature (El-Sheshtawy et al., 2014). The success of AI depends on several factors including the quality of frozen-thawed semen that is being used.

 

Extended sperm is expanding rapidly with a great demand for semen cryopreservation, especially in the cattle industry. Cryopreservation technique allows spermatozoa to be stored indefinitely under -196°C in liquid nitrogen, which is known to cause detrimental effects on the spermatozoa partly during the freeze-thawing process (Watson, 2000). To preserve the semen, cryopreservation media is needed during the cryopreservation process. In order to have a good post-thawed semen quality, the composition of the cryopreservation media used as semen extenders be as one of the most important factors (Yimer et al., 2015). Currently, many studies have been conducted to develop a cheaper and more effective alternatives by using different extenders in the frozen semen which help in extending the fertility of the sperm and reducing sperm damage especially during the cryopreservation stage (Layek et al., 2016). Some researchers have reported the benefits of using honey as a supplementation in the cryopreservation media in various species such as sheep (Yodmingkwan et al., 2016), cattle (El-Sheshtawy et al., 2014), buffaloes (El-Nattat et al., 2016), horses (El-Sheshtawy et al., 2016), and even in human (Fakhrildin and Alsaadi, 2014).

 

The honey bees are bees of the single genus Apis from the tribe of Apini, where the most popular subspecies is Apis mellifera that are found in Europe and Asia. Honey derived from this bee is rich in phenolic compounds that are strongly correlated with antioxidant activity (Kek et al., 2014). Fuller (2004) reported that honey consists of high carbohydrate, protein, amino acids, vitamins, mineral, and composed of antioxidant which helps to prevent the free radicals activity. Besides, honey is a good source of glucose and fructose which help to maintain the osmotic pressure of the diluents by inducing cell dehydration and less formation of ice crystals within and around the spermatozoa, which made it a good additive in frozen semen (Yimer et al., 2015). Nevertheless, the composition of honey depends on the plant species swarmed by the honey bees, the environment, processing, and storage conditions (El-Sheshtawy et al., 2014). To the best of our knowledge, information on honey as an additive in Bioxcell™ extender for cryopreservation is rather limited. Hence, the present study was conducted to determine the effect of using local honey derived from the Apis spp. bee as an additive in Bioxcell™ extender for cryopreservation on bull semen quality from different cattle breeds.

 

Materials and methods

 

Extender Preparation

Commercial extender (Bioxcell™, IMV Technologies, France) was used as control extender. Local honey (Kira Haq®) produced from Apis mellifera bee was used as an additive to the commercial extender. Different concentrations of honey additive in the commercial extenders (1, 2.5, 5, 10 and 15%) were prepared accordingly. The reverse pipetting method was used to measure the volume of honey before been added into the Bioxcel™ extender. The mixtures were mixed well for 10 seconds. All the extenders were placed in a water bath at 37°C for direct usage (El-Sheshtawy et al., 2014).

 

 

 

Animals and Semen Collection

All experimental procedures were conducted in accordance to the Institutional Animal Care and Use Committee of Research Policy at Universiti Putra Malaysia (UPM). Twelve sexually mature bulls consisted of three Mafriwal, Jersey, Peidmontese, and Limousin bulls of each breed were used in the present work (Figure 1-4). Both Mafriwal and Jersey bulls were born and raised locally, while Peidmontese and Limousin bulls were imported from Australia for genetic improvement programs. All bulls were within four to five years of age with body weight ranging from 600 to 800 kg. The bulls were managed intensively which the vaccination and deworming status were up-to-date. All animals were fed with Napier grass as the basal diet using cut and carry system and were supplemented with palm kernel cake based diet. Water was available ad libitum. Semen sample from each bull was collected with the aid of an artificial vagina (AV) and a teaser cow. Ejaculates were collected, stored in insulated jacket, and were immediately transported to the laboratory for further processing and evaluation.

 

 

 

Semen Cryopreservation

After collection, the ejaculates were immediately evaluated for volume, progressive motility, and concentration that was determined by a haemocytometer. Only ejaculates with at least 60% progressively motile sperm and 2.5 × 108 sperm cell/mL were used for freezing. The semen was extended 1:1 (semen: extender) in the prepared extenders that had been warmed to 37°C. The first semen evaluation was carried out on the fresh extended semen. The samples were then chilled at 4°C for two hours in the chiller where the second post-chilled semen evaluation was conducted. The samples were then loaded into 0.25 mL straws with polyvinyl alcohol (PVA) powder and were sealed thermally. Prior to freezing, the straws underwent a pre-freezing phase where the straws were placed on a rack above the liquid nitrogen surface in the freezing vapour container. The samples were gradually frozen in the liquid nitrogen vapour at -120°C for 4 minutes before being immersed into the liquid nitrogen tank at -196°C and stored for two weeks. The third semen evaluation was performed on day 14 where the samples were thawed in a water bath at 37°C for 45 seconds before assessment of the post-thawed frozen semen (Malik, 2018).

 

Semen Evaluation

Evaluations were carried out on fresh semen, post-chilled, and 14th day post-thawed frozen semen. The semen samples were evaluated based on microscopic characteristics such as general motility, progressive motility, and liveability. General motility and progressive motility of the sperm were evaluated by using Computer Assisted Semen Analyser (CASA, IVOS System, Hamilton Thorne Inc., USA). Spermatozoa liveability was assessed using eosin-nigrosin staining technique. One drop of each semen sample was added on top of a slide then mixed with one drop of eosin-nigrosin stain and smeared with a coverslip. A total of 100 spermatozoa were examined under a light microscope (Nikon Eclipse 100 LED Binocular Microscope) for evaluation of spermatozoa liveability, based on their staining characteristics. Spermatozoa that stained pink-purple due to absorption of eosin-nigrosin were considered dead while those remain white were considered viable (Malik, 2018).

 

Statistical Analysis

All data collected were analysed using Statistical Analysis Software (SAS) version 9.4. One-way analysis of variance (ANOVA) and Duncan’s multiple comparison post-hoc tests were used to compare means between treatment groups. The data were considered significant at p<0.05.

 

Results

 

The effect of honey as an additive for cryopreservation on the sperm quality of fresh, post-chilled, and post-thawed frozen bull semen from different cattle breeds are presented in Table 1, 2, and 3 respectively. There were significant differences (P˂0.05) in the sperm quality among different breeds of bull and different concentrations of honey additive extenders.

 

In the first fresh semen evaluation, Jersey bull exhibited the best sperm quality with the highest sperm general motility, progressive motility, and liveability, followed by Mafriwal, Piedmontese, and Limousin bull that demonstrated the lowest result in the control Bioxcell™ extender. The sperm

 

Table 1: The sperm quality of fresh bull semen for control (Bioxcell™), 1%, 2.5%, 5%, 10% and 15% concentration of honey additive extender groups.

 

Fresh Bull Semen (%)
Treatment Bioxcell™ Bioxcell™ + 1% H Bioxcell™ + 2.5% H Bioxcell™ + 5% H Bioxcell™ + 10% H Bioxcell™+ 15% H
General Motility            
Jersey 90.00±1.34ᵅ 91.50±4.50ᵅ 79.80±5.38ᵅ 66.20±4.06ᵇ

59.8±9.18c

9.20±4.42c

Mafriwal 87.20± 5.35ᵅ 87.50±6.34ᵅ 79.60±3.57ᵅ 73.00±3.58ᵅ 61.8±14.09ᵅᵇ

8.20±6.97c

Limousin 64.00±16.00ᵅ 75.50±2.50ᵅ 72.50±3.50ᵅ 64.50±10.50ᵅ 33.50±8.50ᵅᵇ 2.00±0.41ᵇ
Piedmontese 84.00±1.15ᵅ 88.00±2.00ᵅ 79.33±2.84ᵅ 60.33±7.86ᵅ 50.00±11.93ᵅ

4.00±11.79b

Progressive Motility            
Jersey 69.40±4.49ᵅᵇ 76.00±0.00ᵅ 68.20±5.29ᵅᵇ 53.40±5.04ᵅᵇ 45.80±10.37ᵇ

7.00±1.00c

Mafriwal 65.60±4.13ᵅ 70.50±1.50ᵅ 60.60±2.71ᵅ 64.60±3.71ᵅ 38.80±11.40ᵇ

2.40± 1.16c

Limousin 58.00±2.00ᵅ

60.20±2.50ᵅb

52.50±2.50ᵅᵇ 58.00±6.00ᵅ 34.00±3.52ᵅᵇ 0.50±0.05ᵇ
Piedmontese 67.33±6.77ᵅ 67.50±1.50ᵅ 52.00±3.79ᵅ 60.00±2.08ᵅ 46.67±10.48ᵅ

7.67±3.84ᵇ

Liveability            
Jersey 72.25±3.88ᵅ 80.00±16.00ᵅ 71.80±3.89ᵅ 69.25±1.88ᵅᵇ 62.50±1.10ᵇ 58.00±3.89ᵇ
Mafriwal 75.00±2.00ᵅ 76.67±17.11ᵅ 67.00±15.42ᵅᵇ 71.00±17.98ᵅ 67.33±15.02ᵅᵇ 56.00±14.07ᵇ
Limousin 59.00±1.00ᵇ 65.00±6.00ᵅᵇ 61.00±9.00ᵅᵇ 60.00±0.00ᵅᵇ 60.00±0.00ᵅᵇ 53.50±3.50ᵅ
Piedmontese 68.00±3.00ᵅ 74.33±2.84ᵅ 70.00±8.50ᵅ 71.00±7.77ᵅ 63.0±15.01ᵅ 25.33±10.27ᵇ


All values were expressed as mean ± SEM; a, b, c values with superscript within the same row show significant differences at P< 0.05; H = honey.

 

Table 2: The sperm quality of post-chilled bull semen for control (Bioxcell™), 1%, 2.5%, 5%, 10% and 15% concentration of honey additive extender groups.

 

Post-chilled Bull Semen (%)

 

Treatment Bioxcell™ Bioxcell™ + 1% H Bioxcell™ + 2.5% H Bioxcell™ + 5% H Bioxcell™ + 10% H Bioxcell™+ 15% H

 

General Motility            

 

Jersey 89.60±0.86ᵅ 89.80±1.17ᵅ 86.00±2.00ᵅᵇ 77.40±7.17ᵅᵇ 62.40±15.42ᵇ

21.00±10.21c

 

Mafriwal 88.40± 1.96ᵅ 86.50± 5.50ᵅ 81.00±3.62ᵅᵇ 80.80±3.12ᵅᵇ 51.40±13.34ᵇ

19.20±15.08c

 

Limousin 64.50±11.50ᵅ 75.00±5.00ᵅ 70.00±8.00ᵅ 56.00±6.00ᵅ 28.00±3.00ᵅᵇ 12.50±0.27ᵇ

 

Piedmontese 82.00±5.50ᵅ 85.00±5.00ᵅ 61.67±3.84ᵅᵇ 54.33±16.17ᵅᵇ

40.33±11.57bc

16.33±4.33c

 

Progressive Motility            

 

Jersey 69.40±3.61ᵅ 76.00±9.50ᵅ 68.20±4.47ᵅᵇ

54.80±6.72ᵇc

56.00±2.12ᵇc

9.33±6.01ᵈ

 

Mafriwal 67.00±4.44ᵅ 70.50±2.50ᵅ 61.40± 3.10ᵅ 60.40±3.11ᵅ 30.40±10.26ᵇ

7.40±6.66c

 

Limousin 45.00±1.00ᵅ 64.10±3.20ᵅ 54.50±4.50ᵅ 48.00±0.00ᵅ 25.25±2.18ᵇ 2.50±1.78ᵇ

 

Piedmontese 61.67±9.94ᵅᵇ 65.50±2.50ᵅ

47.67±9.28c

54.33±4.80ᵇc

27.67±5.93ᵈ 2.67±0.88ᵉ

 

Liveability            

 

Jersey 68.00±9.43ᵅ 71.50±4.50ᵅ 59.75±4.91ᵅ 64.25±4.55ᵅ 59.75±10.90ᵅ 49.67±4.91ᵅ

 

Mafriwal 69.00±6.50ᵅ 70.30±3.05ᵅ 64.67±2.84ᵅ 67.25±4.37ᵅ 66.33±6.88ᵅ 49.50±6.50ᵅ

 

Limousin 52.00±8.50ᵅ 62.45±2.90ᵅ 60.00±2.00ᵅ 59.50±1.50ᵅ 58.50±8.50ᵅ 46.00±10.26ᵅ

 

Piedmontese 67.67±6.36ᵅ 70.50±5.50ᵅ 64.00±7.00ᵅ 68.00±5.03ᵅ 66.33±2.40ᵅ 40.67±7.13ᵇ

 


All values were expressed as mean ± SEM; a, b, c, d, e values with superscript within the same row show significant differences at P< 0.05; H = honey.

 

quality of all bull species increased significantly in 1% concentration of honey additive extender. Nonetheless, 2.5%, 5%, 10%, and 15% concentration of honey additive extenders did not yield positive results as the sperm general motility, progressive motility, and liveability decreased significantly as the concentration of honey increased (Table 1).

 

Table 3: The sperm quality of post-thawed frozen bull semen for control (Bioxcell™), 1%, 2.5%, 5%, 10% and 15% concentration of honey additive extender groups.

 

Post-thawed Frozen Bull Semen (%)  
Treatment Bioxcell™ Bioxcell™ + 1% H Bioxcell™ + 2.5% H Bioxcell™ + 5% H Bioxcell™ + 10% H Bioxcell™+ 15% H  
General Motility              
Jersey 52.75±13.08ᵅᵇ 61.50±8.50ᵅ

31.50±11.09ᵇc

30.00±4.91ᵇ

15.67±2.40c

2.00±0.00ᵈ  
Mafriwal 37.00±13.45ᵅ 49.00±11.00ᵅ

19.80±8.95ᵅᵇc

31.20±15.70ᵅᵇ

7.80±3.60ᵇc

1.40±1.16c

 
Limousin 14.00±3.00ᵅ 18.50±4.50ᵅ 8.00±1.72ᵅ 12.50±2.69ᵅ 2.00±0.43ᵅ 3.00±2.15ᵅ  
Piedmontese 31.67±13.11ᵅᵇ 36.50± 7.50ᵅ

8.00±0.58ᵇc

6.67±3.33ᵇc

5.00±3.05ᵇc

0.33±0.24c

 
Progressive Motility              
Jersey 37.5 ± 10.00ᵅ 44.50±9.50ᵅ 23.00±9.30ᵇ

18.00±5.49ᵇc

6.00±1.53cᵈ 1.00±0.43ᵈ  
Mafriwal 20.40±8.86ᵅ 22.00±2.00ᵅ

12.60±6.38ᵅᵇc

14.60±6.28ᵅᵇ

5.40±1.86ᵇc

0.40±0.40c

 
Limousin 12.00±4.35ᵅ 15.00±2.35ᵅ 8.00±2.50ᵅ 8.00±2.50ᵅ

2.20±0.20c

0.10±0.05c

 
Piedmontese 22.67±11.32ᵅ 16.50±14.50ᵅ 3.00±2.08ᵅᵇ 6.67±1.67ᵅᵇ 4.67±1.85ᵅᵇ 1.00±0.58ᵇ  
Liveability              
Jersey 56.50±16.50ᵅ 69.50±6.25ᵅ 56.00±16.00ᵅ 52.00±22.00ᵅ 50.50±10.50ᵅ 45.00±5.00ᵅ  
Mafriwal 53.00±8.33ᵅ 63.50±1.50ᵅ 48.00±8.54ᵅᵇ 49.67±6.36ᵅᵇ 46.33±13.59ᵇ 45.00±6.08ᵅᵇ  
Limousin 40.00±14.50ᵅᵇ 49.00±15.00ᵅ 24.00±5.13ᵅᵇ 19.33±11.28ᵅᵇ 22.67±6.69ᵅᵇ 11.67±3.71ᵇ  
Piedmontese 51.00±7.11ᵇ 65.00±10.45ᵅ 52.50±7.31ᵇ 52.00±8.64ᵇ

27.50±3.83c

44.00±6.13ᵇc

 


All values were expressed as mean ± SEM; a, b, c, d values with superscript within the same row show significant differences at P< 0.05; H = honey.

 

Similar results were observed in the second post-chilled bull semen evaluation where Jersey bull and Limousin bull has the highest and lowest sperm quality values correspondingly. 1% concentration of honey additive extender was able to increase the sperm quality after chilling at 4°C for 2 hours in the chiller compared to the control extender. On the other hand, other concentrations of honey additive extenders were unable to preserve the sperm quality as the values were significantly lower than the control and 1% concentration of honey additive extenders (Table 2).

 

The third semen evaluation of post-thawed frozen bull semen showed a significant reduction in the sperm general motility, progressive motility, and liveability in all the bulls’ semen with Limousin breed exhibiting the worst sperm quality. Although Jersey bull semen revealed the highest figures of semen quality after thawing, all the values were below 60% in the control, 2.5%, 5%, 10%, and 15% concentration of honey additive extenders. In contrast, 1% concentration of honey additive extender was able to maintain a better quality of sperm with higher general motility, progressive motility, and liveability in comparison to the other extenders (Table 3).

 

Discussion

 

The successful outcome of AI depends on the semen quality which is being influenced by many factors such as management, nutritional factors, breeds, and also seasonal variation. According to Kastelic (2013) Bos taurus bulls are more susceptible to high ambient temperatures than Bos indicus bulls. In the current work, Jersey (a Bos taurus breed) and Mafriwal (a synthetic breed) exhibited a better sperm quality, as compared to Piedmontese and Limousin (both are Bos taurus breed). This could be due to the environmental adaptation of these two breeds which were bred and raised locally. Locally bred animals may show a genetic predisposition to heat tolerance comparing to foreign breeds (Rojas-Downing et al., 2017). In contrast, both Piedmontese and Limousin bulls were imported from Australia where the differences in the management, nutrition, and environmental factors affect the animals’ fertility. The hot and humid tropical weather in Malaysia could be one of the main factors affecting the performance of these imported bulls. Heat stress is often worsened in the tropical region due to the excessive humidity which causes livestock to feel hotter than the actual temperature (Chung et al., 2019). This will consequently affect the physiological performance of cattle with the most affected is the decrease in fertility. Nevertheless, the success of AI also depends on the sperm quality based on their performance in motility, viability, abnormality, and concentration of sperm, especially for the semen which will be used for cryopreservation (El-Sheshtawy et al., 2014). As shown in this study, Jersey bull demonstrated the best overall sperm quality based on the fresh semen, post-chilled, and 14th day post-thawed frozen semen evaluations, achieving the minimum standard for health and structural soundness (Kastelic, 2013). The results were consistent with Kumar et al. (2015), which concluded that the fresh, pre-freeze, and post-freeze semen quality of Jersey bulls were comparatively better than the crossbred Jersey bulls. Thus, the selection of cattle breed in a particular region plays an important role in producing good bull semen quality for cryopreservation and AI.

 

Cryopreservation of cattle semen is the most commonly practiced compared to other livestock. Semen will be collected and evaluated to determine the breeding potential before it is being preserved (Bhattacharyya et al., 2009). With this technology improvement, semen from good bulls can be kept or cryopreserved for many years by stabilizing the cells at the cryogenic temperatures (Fakhrildin and Alsaadi, 2014). The performance rate of individual bull might influence the successfulness of cryopreservation. This was shown in the present study, where the bulls that showed better sperm quality in the fresh semen will have better general motility, progressive motility, and liveability during the thawing stage. There are two major damaging events identified during cryopreservation which are freeze-dehydration, that cause changes in the ultrastructure of the cell membrane that lead to organelle disruption, and the second major disruption is the propagation of intracellular ice crystal (Fuller, 2004). This cryoinjury usually caused by the intracellular ice formation and also by the solution or cryopreservation media effects (Baust et al., 2009). Furthermore, there are three phases that can cause progressive cell injury on the spermatozoa which are cooling, maintenance in the cold, and rewarming. Based on previous research, 40-50% of the sperm do not survive cryopreservation after thawing, even with optimised protocols (Bansal and Bilaspuri, 2011; Lyashenko, 2015). The researchers also stated that, after thawing, the results for spermatozoa are generally poorer than fresh semen which was observed in the present work during the post-thawed frozen bull semen evaluation. Hence, the addition of cryoprotectant agent is crucial in order to prevent the spermatozoa from freezing damage (Aisen et al., 2005; Yoon et al., 2016).

 

Cryoprotectant helps in protecting the sperm cells and yield a higher post-thaw survival rate (Raheja et al., 2018). It helps to maintain spermatozoa’s requirements, control the pH changes in the extracellular environment of the spermatozoa, minimizing cryogenic damage, and also as a contamination control (Yimer et al., 2015). Cryoprotectant agent can be classified as non-penetrating cryoprotectant and penetrating cryoprotectant. One of the contents in penetrating cryoprotectant is sugar which consists of glucose, lactose, raffinose, saccharose, and trehalose (Purdy, 2006). Addition of sugar in semen extender helps to provide energy to the sperm cells, which results in increased fertility of the sperm (Aires, 2003). Honey is mainly composed of 79.6 % of sugars and 17.2% of water which established honey as one of the simple sugar sources which might serve both as a source of nutrition and non-penetrating cryoprotectant to sperm cells during cryopreservation (Khalil et al., 2010; Jerez-Ebensperger et al., 2015). Conferring to this study, 1% concentration of honey was suitable to be added in Bioxcell™ extender as an additive to give a better performance in all bull semen quality at different sperm cryopreservation phases. This finding was concurrent with Malik (2016) reported that honey solution addition in different semen extenders improved the sperm motility in both chilled and frozen semen. Honey is rich in phenolic compounds that are related to the strong antioxidant activity as it contains a mixture of carbohydrates, proteins, enzymes, amino acids, and organic acids, vitamins, phenolic acids, and flavonoids (Kek et al., 2014). So, sufficient nutrients present in the honey additive extender increase the metabolic activity of the spermatozoa and lead to a lower number of dead spermatozoa due to the strong antioxidant property.

 

Naturally, honey is a highly concentrated product and has the potential to cause a hyperosmotic extracellular environment around sperm cells. This will enhance the efflux of intracellular fluid which in turn minimizing ice crystallization in the sperm cytoplasm that causes damage during cryopreservation (Pegg, 2010; Erejuwa et al., 2012). For that reason, too high concentration of honey will also affect the fertility of the spermatozoa due to the changes in pH, osmolarity, and sugar level. Our data have proven that the addition of 2.5, 5, 10, and 15% concentration of the honey additive in Bioxcell™ extender revealed significantly poor sperm quality during semen evaluation, especially at post-cryopreservation. Addition of high concentrations of honey especially 15% of honey was not suitable as an additive which causes osmotic stress that reduces the semen quality. Osmotic stress that occurs will change the membrane organisation and altered membrane fluidity and permeability which compromised the sperm function (Watson, 2000). The used of high concentration of non-penetrating cryoprotectant will cause a hyperosmotic extracellular environment for the spermatozoa leading to excessive intracellular dehydration (Purdy, 2006). As a result, too high concentration of honey as an additive in Bioxcell™ extender was not suitable and resulted in poor bull semen quality.

 

Conclusion

 

In conclusion, the present experiment showed that there were significant differences in the bull semen quality between breeds and between different concentrations of honey additive extenders. Jersey bull exhibited the best sperm quality compared to Mafriwal, Piedmontese and Limousin bull. The general motility, progressive motility, and liveability of sperm in the 1% concentration of honey additive extender were improved and preserved throughout the semen cryopreservation process compared to 2.5, 5, 10 and 15% concentration of honey additive extenders. Therefore, 1% concentration of honey additive extender could be effectively used in bull semen cryopreservation.

 

Acknowledgement

 

The authors would like to thank the staff from Institut Biodiversiti Veterinar Kebangsaan Malaysia (IBVK) for their technical assistance.

 

Conflict of interest

 

The authors declare that they have no conflict of interest.

 

authors contribution

 

All authors contributed equally and approved the final manuscript.

 

References

 

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