Effect of Bioxcell and Triladyl Extenders and Removal of Seminal Plasma on Equilibrated and Cryopreserved Semen from South African Unimproved Indigenous Bucks

Research Article

J Bacteriol Mycol. 2021; 8(1): 1164.

Effect of Bioxcell® and Triladyl® Extenders and Removal of Seminal Plasma on Equilibrated and Cryopreserved Semen from South African Unimproved Indigenous Bucks

Nethenzheni LP¹*, Mphaphathi ML², Madzhie LR¹, Negota NC¹, Kalonji PVM¹, Nedambale TL³ and Barry DM¹

1Center of Excellence in Animal Assisted Reproduction, University of Venda, South Africa

2Agricultural Research Council, Animal Production Institute, South Africa

3Department of Animal Sciences, Tshwane University of Technology, South Africa

*Corresponding author: Livhuwani Pertunia Nethenzheni, Center of Excellence in Animal Assisted Reproduction, University of Venda, School of Agriculture, Private Bag X 5050, Thohoyandou, 0950, Republic of South Africa

Received: January 25, 2021; Accepted: February 17, 2021 Published: February 24, 2021

Abstract

The objectives of the study were to evaluate the effect of two extenders (Triladyl® and Bioxcell®) and the removal of seminal plasma on indigenous buck’s semen. Semen was collected from six indigenous bucks using an electro-ejaculator. Raw semen was pooled and randomly allocated into six groups as follows: (i) Raw non-washed, (ii) Raw washed, (iii) Triladyl®-washed, (iv) Triladyl®-non-washed, (v) Bioxcell®-washed and (vi) Bioxcell®-non-washed. Both the Triladyl® and Bioxcell® washed semen samples groups were diluted (1:4 v/v) with Phosphate Buffered Saline (PBS) then centrifuged at 1500x g for ten min and seminal plasma was removed. The groups were analysed for spermatozoa motility rates using Computer-Aided Sperm Analysis (CASA). The spermatozoa viability was assessed using Eosin-Nigrosin, acrosome integrity using Spermac, chromatin structure using Acridine Orange, and mitochondria using JC-1 staining solutions. Semen samples were diluted (1:4 v/v) as follows: Triladyl® (washed and non-washed) or Bioxcell® (washed and non-washed) and then equilibrated at 5°C for 2 hours. Equilibrated semen samples in 0.25 mL French straws were placed 5 cm above a Liquid Nitrogen (LN2) vapour for 10 min, and stored for one month. Frozen semen straws per treatment group were thawed at 37°C for 30 seconds. Significant differences among the mean values of semen parameters were determined by Tukey’s test using ANOVA, GLM procedure of SAS version 12.1 of 2010. The spermatozoa progressive motility rate in non-washed semen extended with Bioxcell® was significantly higher (89.6±7.5a) compared with that of non-washed Triladyl®, washed Bioxcell® and Triladyl® (P<0.05). Live spermatozoa percentage in washed semen extended with Triladyl® extender was reduced (27.7±17.1) significantly compared with the other groups (P<0.05). There was a lower percentage of spermatozoa with high mitochondrial membrane potential in non-washed and washed semen extended with Bioxcell® (39.5±23.2 and 37.9±28.6, respectively) compared with that of non-washed and washed semen extended with Triladyl® (P>0.05). The spermatozoa progressive motility rate in non-washed semen extended with Bioxcell® (58.5±10.0) extender was significantly higher compared with that of the other groups (P<0.05). There was a higher live and normal spermatozoa percentage in non-washed semen extended with Bioxcell® (45.7±21.2) compared with that of the other groups (P<0.05). In conclusion, Washing of seminal plasma in semen extended with Triladyl® was not essential, as it lowered viability, progressive motility and chromatin membrane integrity prior and post-cryopreservation. However, Bioxcell® extender was found to be more suitable for preserving spermatozoa during equilibration and freezing/thawing process of non-washed and washed buck semen.

Keywords: Indigenous Bucks; Seminal Plasma; Membrane Integrity; Bioxcell®; Triladyl®

Introduction

Goats are prolific and require low inputs for a moderate level of production. They tend to reach maturity early and are profitable to keep [1]. Also, goats are tolerant of different parasites and diseases, non-selective browsers and able to survive on the poorest vegetation [2]. Most South African indigenous goats are kept in rural areas and used for milk, hides, and meat production [1]. However, there is limited information regarding their reproductive status [2], semen quality and their tolerance to cryopreservation process [3]. This is mainly because they have received little attention from researchers/ scientist in the past. However, there is recent interest from commercial farmers and this may be due to their hardiness and adaptability to the local harsh environmental conditions as well as their exceptional capacity to produce and reproduce efficiently under poor nutritional conditions [3].

Furthermore, there is a need to conserve their genetic materials. Genetic resource banks are normally utilised with reproductive technologies for the conservation of endangered species [4]. Semen collection and storage is essential in controlled breeding programs of many endangered species [5]. The cryopreservation of gametes is vital because it would allow researchers to support a genome resource bank for this breed for an indefinite period [6]. Cryopreservation of goat spermatozoa also extends the male reproductive life of a goat germline [7].

Frozen-thawed goat semen may be utilised for Artificial Insemination (AI) to enhance improvement of livestock as breeders mostly make use of genetically superior goats [2]. But, cryopreservation of goat semen remains a challenge due to poor post-thaw sperm motility and viability recovery percentages. This method includes temperature reduction, cellular dehydration, eventual freezing and subsequent thawing. Cryopreservation has been shown to stop all cellular activities, restarting its normal metabolic functions, following thawing. Nevertheless, semen cryopreservation generally induces the formation of intracellular ice crystals, osmotic and chilling injury, which causes sperm cell damage, cytoplasmic fracture, or some effects on the cytoskeleton or the genome-related structures [8-10]. These eventually impair spermatozoa transport and decrease the survival rate in the female reproductive tract, thus reducing fertility [10].

Phospholipase A2 activity of Egg Yolk Coagulating Enzyme (EYCE) catalyses the hydrolysis of egg yolk Phosphatidylcholine (PC) into fatty acids and Lysophosphatidylcholine (LPC). The LPC has a toxic effect on buck spermatozoa when it is extended with eggyolk- based extenders by acting like a detergent on biomembrane, resulting in a loss of motility, membrane integrity and consequently low fertility rate [11,12]. Among the factors affecting the freezing ability of spermatozoa, supplementation of the freezing medium with different types of cryoprotectant plays a vital role in minimising the physical and chemical stresses occurring during cryopreservation procedure [13].

Hence, it is of utmost importance to develop and modify protocols to improve cryopreservation protocols to preserve indigenous germplasm. Seminal plasma plays an important role in spermatozoa survival during the cryopreservation process. This is because the causes of reduced spermatozoa motility are related to seminal plasma enzymes [11]. Goat semen is currently washed to eliminate the seminal plasma from the spermatozoa prior to dilution with standards extenders containing egg yolk [14]. Currently, soylecithin- based extenders are used to avoid problems encountered when egg-yolk-based extenders are used to process goat semen [15]. The present study focused on the effect of extenders (egg-yolk-based extender and soy-lecithin-based extender) and the removal of seminal plasma on South African indigenous goat semen parameters.

Materials and Methods

Chemicals

The extenders used in this study were Bioxcell® (IMV, L’Aigle and France) and Triladyl® (Minitüb, Germany). The stains used were Spermac® stain (Stain enterprises, South Africa), Acridine Orange (AO), Phosphate Buffered Saline (PBS) and Dimethyl Sulfoxide (DMSO) (Sigma-Aldrich, St Louis, MO, USA), JC-1 stain (Molecular probesTM, Eugene. Oregon. USA), and Eosin-Negrosin (Merck Millipores corporation, South Africa).

Experimental bucks, semen collection and processing

The study was conducted at the University Of Venda Centre Of Excellence in Animal Assisted Reproduction (CEAAR). Semen samples were collected twice a week (spring to early summer) from six unimproved indigenous bucks (3 to 4 years of age). They were kept in pens and maintained on ewe and lamb pellets with water provided ad libitum throughout the experiment. Semen was collected with the aid of an electro-ejaculator into pre-warmed (37°C) 15 mL graduated plastic tubes and immediately placed in a thermo flask with water at 37°C. The collected buck semen samples were then transported to the laboratory for further analysis.

The buck semen samples were pooled to eliminate individual differences between the samples. Pooled semen was randomly allocated into six aliquots, namely (i) Raw non-washed, (ii) Raw washed, (iii) Triladyl®-washed semen, (iv) Triladyl®-non-washed semen, (v) Bioxcell®-washed semen and (vi) Bioxcell®-non-washed washed. Triladyl® and Bioxcell® washed semen groups were mixed with PBS at a ratio of 1:4 v/v. Semen samples were then centrifuged at 1500x g for 10 min. Following centrifugation, seminal plasma was removed using 1mL sterile plastic hand pipette, leaving only the spermatozoa pellets.

Preparations of semen extenders

Preparation of Bioxcell® extender: One bottle of Bioxcell® (100mL) was diluted with 400mL of distilled water for a final extended, ready to use volume of 500mL. It was then aliquoted into 15mL graduated tubes and stored in the refrigerator (-20°C) until use.

Preparation of Triladyl® extender: The chicken egg yolk was separated from the albumen by passing the egg yolk from onehalf of the shell to the other to get rid of the albumin [7]. The egg yolk was placed in a gauze swab and an 18-gauge needle was used to punch the egg yolk to separate the egg yolk from its membrane and some contents of albumin to the 15 mL graduated tube. Triladyl® concentrate was added slowly to the water and the mixture was added to egg yolk. The mixture was stirred and filtered using a sterile filter funnel. The final mixture of Triladyl® extender was kept in 15 mL graduated tubes, sealed with Parafilm and stored in the refrigerator (at 5°C).

Semen extension, equilibration, freezing and thawing

Semen samples were extended with pre-warmed (37°C) Bioxcell® or Triladyl® at a ratio of 1:4 v/v (semen to extender). Rapid cooling was done by placing the tubes in a beaker with cool water at room temperature (25°C). After 10 min, the beaker was transferred into the refrigerator and equilibrated at 5°C for two hours [7]. After equilibration, the semen samples were loaded into 0.25 mL French straws and sealed with polyvinyl powder per treatment group. The straws were frozen in LN2 (-196°C), 5 cm above a liquid nitrogen surface for 10 min and plunged into LN2 and stored for a month.

Semen evaluation

Evaluation of spermatozoa motility: Spermatozoa motility rate was analysed using the Computer-Aided Sperm Analyser (CASA) system, version 5.4, (Microptic SL, Barcelona, Spain) [7]. Five hundred microliters (μL) of PBS plus 20 μL of raw semen were mixed in an Eppendorf tube using a hand pipette. A drop (2 μL) of semen sample was placed on a pre-warmed (37°C) microscopic glass slide and mounted with a cover of a microscopic glass slip and examined under a CASA microscope. Four fields per sample were captured under 10X magnification with a Ph1 phase contrast.

Evaluation of spermatozoa viability (live/dead): Spermatozoa viability was analysed using an Eosin-Nigrosin staining solution [16]. A drop of 20 μL Eosin and a drop of 10 μL Nigrosin were placed on the one end of the pre-warmed (37°C) microscopic glass slide using a hand pipette. Then 10 μL of semen was placed on the stains and they were gently mixed together with the tip of the hand pipette and then a smear was made using a pipette tip (to avoid breakage of spermatozoa during the smear) at 37°C. The slide was placed on the slide warmer at 120°C to allow the smear to dry fast. Dried microscopic slides were analysed for viability using the CASA microscope at 60X magnification with oil immersion and 300 spermatozoa were counted/slide. The live spermatozoa are unstained and the dead spermatozoa are stained.

Evaluation of spermatozoa acrosome membrane damage: Spermatozoa acrosome membrane damage was analysed by staining spermatozoa with Spermac® stain [16]. A drop of 15 μL of semen was placed on a microscopic glass slide using a hand pipette. A smear was made by gently pulling semen from one end to the other end using a hand pipette tip and allowed to air dry for about 10 min on a warm plate at 37°C. The absorbent paper was spread inside the staining tray to absorb excess stain during the staining procedure and the smeared slide was placed on the staining tray. The smeared slide was fixed with the Spermac® fixative by placing fixative drops on the smeared slide until it was covered with the fixative using a hand pipette. After 1 min, the microscopic slide was then placed vertically on absorbent paper to drain excess fixative into the staining tray. The slide was then washed gently by dipping it seven times in a glass beaker with distilled water to remove the stain. The fixed slide was placed on the staining tray with absorbent paper and stained with stain A by placing stain drops on the fixed slide until it was covered with stain A using the hand pipette for 1 min. The stained slide was then drained by holding the slide vertical on its side and washed by dipping the slide seven times in a glass beaker with distilled water. The slide was placed vertically on absorbent paper to drain excess water. The same procedures were repeated with stains B and C. The slides were air dried for 15 min and then observed under a light microscope (60X magnification) using oil immersion and 300 spermatozoa per stained slide were counted. The acrosome stained dark green and nucleus stained red.

Acridine Orange (AO) staining procedure for evaluation of sperm chromatin structure: The AO staining method was from a protocol previously described by [17]. The raw semen sample was washed three times in PBS by centrifuging at 1500x g for 10 min. Fifteen microliter drops of the spermatozoa pellets were smeared on a microscopic slide using a hand pipette tip and allowed to air-dry for 10 min. The smears were fixed for 1 hr in ethanol-acetone (1:1) at 4°C and allowed to air-dry for 10 min. The microscopic slides were then stained for 7 min with AO (0.19 mg/mL) at room temperature in the dark. The slides were gently rinsed with distilled water and air-dried for 10 min, then viewed under a UV light inverted microscope at 40X magnification (Nikon eclipse TI, Narishige Co., Ltd. USA). Three hundred spermatozoa per stained slide were counted.

JC-1 staining procedure for evaluation of sperm mitochondrial membrane potential: The JC-1 stain was used to analyse the spermatozoa mitochondrial membrane potential [18]. Five mg of JC-1 was dissolved in 5 mL of DMSO. Semen was diluted with PBS (1:4 v/v) and centrifuged at 1500 x g for 10 min and the fluid was discarded. Ten microliters of JC-1 staining solution was diluted in pre-warmed 100 μl of PBS in an Eppendorf tube and placed in a water bath at 37°C. The spermatozoa pellets were diluted with PBS and 5 μL of PBS with spermatozoa were mixed with 5 μl of the JC-1 staining solution in an Eppendorf tube and incubated for 10 minutes at 37°C. Incubated spermatozoa were washed by mixing with 40 μL of PBS using a hand pipette. Two microliters of PBS plus spermatozoa was placed on a slide and covered with a coverslip. Spermatozoa were observed immediately under a UV light inverted microscope at 40X magnification (Nikon eclipse TI, Narishige Co., Ltd. USA). Three hundred spermatozoa per stained slide were counted. The spermatozoa mitochondria with high membrane potential appeared reddish and mitochondria with low membrane potential appeared greenish.

Statistical analysis

The data were Analysed by Analysis of Variance (ANOVA) for a 3 X 2 factorial design in a completely randomized design using the General Linear Model (GLM) procedure of SAS version 12.1 of 2010. A significant difference (P<0.05) among the mean values of semen parameters were determined by Tukey’s test method.

Results

Sperm motility, viability, acrosome, chromatin and mitochondrial membrane potential of equilibrated semen

The spermatozoa total motility rate in non-washed semen extended with Bioxcell® was significantly higher (97.8±2.3) compared with that of non-washed Triladyl® and washed Bioxcell® extended semen (P<0.05) (Table 1). The spermatozoa progressive motility rate in non-washed semen extended with Bioxcell® was significantly higher (89.6±7.5a) compared with that of non-washed Triladyl®, washed Bioxcell® and Triladyl® (P<0.05). Live spermatozoa percentage in washed semen extended with Triladyl® extender was reduced (27.7±17.1) significantly compared with the other groups (P<0.05). The percentage of spermatozoa acrosome integrity in non-washed semen extended with Bioxcell® (71.1±13.8) extender was not significantly different from the other groups (P<0.05). The spermatozoa chromatin integrity was lower in washed semen extended with Triladyl® (46.3±9.4) compared with non-washed semen extended with Bioxcell® and Triladyl® extenders (P>0.05). There was a lower percentage of spermatozoa with high mitochondrial membrane potential in non-washed and washed semen extended with Bioxcell® (39.5±23.2 and 37.9±28.6, respectively) compared with that of nonwashed and washed semen extended with Triladyl® (P>0.05).

Citation: Nethenzheni LP, Mphaphathi ML, Madzhie LR, Negota NC, Kalonji PVM, Nedambale TL, et al. Effect of Bioxcell® and Triladyl® Extenders and Removal of Seminal Plasma on Equilibrated and Cryopreserved Semen from South African Unimproved Indigenous Bucks. J Bacteriol Mycol. 2021; 8(1): 1164.