The objectives of the study were to evaluate the effect of two extenders (Triladyl® and Bioxcell®) and the removal of seminal plasma on goat semen. PAGE Table 3.1 Composition of Bioxcell® and Triladyl® semen extender 28 Table 4.1 Effect of extenders (Bioxcell® & Triladyl®) and seminal plasma (non-washed and washed).
- Background
- Problem statement
- Justification
- Objective of the study
- The specific objectives are to
- Hypotheses
Goat semen is currently centrifuged (washed) to remove the seminal plasma from the sperm before dilution with standard fillers containing egg yolk. The effect of seminal plasma on goat semen fertility as measured by semen parameters during cryopreservation should be investigated.
- Introduction
- Description of semen
- Spermatogenesis
- Hormonal control of spermatogenesis
- Follicle stimulating hormone (FSH) or spermatogenesis stimulating hormone
- Luteinizing hormone (LH) or interstitial cell stimulating hormone (ICSH)
- Testosterone
- Spermatozoa structure and function
- Plasma membrane
- Spermatozoa head
- Spermatozoa mitochondrion
- Seminal plasma
- Effect of cryopreservation on the spermatozoa structure and functions
- Factors affecting semen production and quality
- Semen collection methods
- Semen collection using the artificial vagina
- Semen collection by electro-ejaculation
- Semen evaluation
- Manual/visual spermatozoa analysis
- Automatic spermatozoa analysis procedures
- Evaluation of semen parameters
- Semen colour and volume
- Semen pH
- Semen concentration
- Spermatozoa motility
- Spermatozoa morphology
- Cryopreservation of semen
- Cryoprotectants used in semen cryopreservation
- Antioxidants
- Antibiotics
- Buffers
The functions of the testes are to produce spermatozoa and androgens (testosterone), regulated by specific hormones (follicle-stimulating hormone (FSH) and luteinizing hormones (LH)). Follicle-stimulating hormone is one of the gonadotropins (GTHs) produced in the pituitary gland, which are members of the pituitary glycoprotein family. The acrosome is a membrane-enclosed structure located on the head of the spermatozoa (Lida et al., 1999; Hossain et al., 2011).
During the passage of the spermatozoa through the epididymis, the protamines formulate inter- and intra-molecular disulfide bonds, and as a result spermatozoa chromatin is fully condensed (Garcia-Macias et al., 2006). Boar spermatozoa adhesins are a group of proteins found in seminal plasma and are major secretory products of the seminal vesicle epithelium (Teixeira et al., 2006). The ROS have been shown to alter cellular functions by disrupting the spermatozoa plasma membrane and damaging proteins and DNA (Hammadeh et al. 1999; Memon et al., 2012; Lee et al., 2014).
The presence of blood in the semen is indicated by a pink color of the semen (contamination) and may be due to injury or disease of the penis or reproductive system (Matshaba, 2010; Munyai, 2012). A pH of about 6.8 to 7.0 is within the optimal range of activity for most of the enzymes in the spermatozoa. Morphological assessment of the spermatozoa is an integral part of semen analysis and is an important part of any study of the soundness of the breeding goat (Mekasha et al., 2007; Matshaba, 2010).
Tertiary abnormalities are the result of poor handling of the semen after collection (reacted acrosomes and coiled spermatozoa tails (Ramukhithi, 2011). The assembly of the extenders helps to stabilize the cell during the freezing and thawing process (Soylu et al., 2007).
- Study site
- Experimental bucks
- Semen collection and processing
- Semen evaluation before extension
- Evaluation of spermatozoa motility
- Evaluation of spermatozoa viability (live/dead)
- Evaluation of spermatozoa morphology and acrosome membrane damage
- Acridine orange (AO) staining procedure for evaluation of chromatin structure.26
- Preparation and composition of semen extenders
- Semen extension, equilibration and evaluation of semen samples
- Freezing of semen
- Semen thawing procedure
- Statistical analysis
A drop of 20 µL Eosin and a drop of 10 µL Nigrosin were placed on one end of the pre-warmed (37 °C) microscope slide (MS labcon) using a hand pipette. Fifteen microliter drops of spermatozoa pellets were smeared on a microscope slide using a hand pipette tip and allowed to air dry for 10 minutes. To avoid repeated freeze/thaw of the JC-1 stock solution, small aliquots were made after the first thaw and stored at -20°C.
A hand pipette was used to gently aspirate the fluid to avoid disturbing the spermatozoa. 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. The yolk was separated from the egg white by passing the yolk from one half of the shell to the other to get rid of the albumin (Ajao, 2015).
At the end of the 10 minutes, all semen straws were immersed directly in the LN2 (-196 ºC). Egg is the effect of the iste extender. Pj is the effect of the jth plasma status. EP)ij is the interaction between the ith extender and the jth plasma status (ES)ik is the interaction between the ith extender and the kth semen status. EPS)ijk is the effect of the interaction of the ith extender jth plasma status kth semen status.
The effect of extenders and removal of seminal plasma on semen parameters of
The percentage of sperm velocity in a straight line (VSL) was significantly higher (P < 0.05) in unwashed sperm elongated with Bioxcell extender than sperm elongated with Triladyl extender. The percentage of VSL spermatozoa was significantly higher (P < 0.05) in washed sperm extended with Bioxcell extender than in sperm extended with Triladyl extender. Mean sperm path velocity (VAP) was significantly higher (P < 0.05) in unwashed sperm elongated with Bioxcell extender than sperm elongated with Triladyl extender.
Raw NW = raw not washed, Biox NW 2 hours = Bioxcell® not washed 2 hours, Vibrate NW 2 hours = Triladyl® not washed 2 hours, Raw W = raw washed, Biox W 2 hours = Bioxcell® washed 2 hours, Vibration W 2 h = Triladyl® washed 2 h, VCL = curve speed, VSL = straight line speed, VAP = average web speed, LIN = linearity, STR = straightness, WOB = wobbling, ALH = amplitude of lateral displacement of the head, BCF = beat cross frequency. There was no significant difference (P > 0.05) in abnormal morphology of live spermatozoa from sperm samples extended with both Bioxcell® and Triladyl® extenders from unwashed and washed sperm samples. The percentage of acrosome integrity of spermatozoa in unwashed sperm extended with Bioxcell extender was not significantly different (P < 0.05) from the sperm extended with Triladyl extender (Table 4.4).
There was a lower (P > 0.05) percentage of spermatozoa with high mitochondrial membrane potential in unwashed sperm extended with the Bioxcell extender than the sperm extended with the Triladyl extender. There was a lower (P > 0.05) percentage of spermatozoa with high mitochondrial membrane potential in washed sperm elongated. Raw NW = raw unwashed, Biox NW 2 hours = Bioxcell® unwashed 2 hours, Tril NV 2 hours = Triladyl® unwashed 2 hours, RåW = raw washed, Biox W 2 hours = Bioxcell® washed 2 hours, Tril W 2 h = Triladyl® washed 2 hours, Δψmhigh = High mitochondrial membrane potential, Δψmlow = Low mitochondrial membrane potential.
The effect of extenders and removal of seminal plasma on semen of South African
35 Table 4.5: Effect of extenders (Bioxcell® & Triladyl®) and seminal plasma (unwashed & . washed) on frozen-thawed sperm motility parameters (mean ± S.E.). Raw NW = raw unwashed, Biox NW FT = Bioxcell® unwashed frozen-thawed, Tril NW FT = Triladyl® unwashed frozen-thawed, Raw W = raw washed, Biox W FT = Bioxcell® washed frozen-thawed, Tril W FT = Triladyl® washed frozen-thawed, TM = total motility, PM = progressive motility, NPM = non-progressive motility, RAP = fast, MED = medium, SLW = slow, STC = static. Raw NW = raw unwashed, Biox NW FT = Bioxcell® unwashed frozen-thawed, Tril NW FT = Triladyl® unwashed frozen-thawed, Raw W = raw washed, Biox W FT = Bioxcell® washed frozen-thawed, Tril W FT = Triladyl® washed frozen-thawed, VCL = curve line speed, VSL = straight line speed, VAP = average road speed, LIN = linearity, STR = straightness, WOB = wobble, ALH = amplitude of lateral head displacement, BCF = beat crossover frequency.
There was a higher (P < 0.05) percentage of spermatozoa with head defects in unwashed sperm extended with Triladyl extender than in the sperm extended with Bioxcell extender. There was a higher (P < 0.05) percentage of spermatozoa with head defects in washed semen samples extended with Triladyl Extender than the sperm extended with Bioxcell Extender. The percentage of spermatozoa with twisted tail abnormalities was significantly reduced (P < 0.05) in washed sperm extended with Triladyl extender compared to the sperm extended with Bioxcell extender.
Raw NW = raw non-washed, Biox NW FT = Bioxcell® non-washed frozen-thawed, Tril NW FT = Triladyl® non-washed frozen-thawed, Raw W = raw washed, Biox W FT = Bioxcell® washed frozen-thawed , Tril W FT = Triladyl® wash freeze-thaw. There was a lower percentage (P < 0.05) of spermatozoa with high mitochondrial membrane potential in unwashed semen extended with Bioxcell extender than the semen extended with Triladyl extender. Raw NW = raw non-washed, Biox NW FT = Bioxcell® non-washed frozen-thawed, Tril NW FT = Triladyl® non-washed frozen-thawed, Raw W = raw washed, Biox W FT = Bioxcell® washed frozen-thawed , Tril W FT = Triladyl® washed freeze-thawed, Δψmhigh = High mitochondrial membrane potential, Δψmlow = Low mitochondrial membrane potential.
The effect of extenders and removal of seminal plasma on semen of South African
The total motility of washed sperm samples extended with Triladyl® was higher than that of Bioxcell® extended sperm samples. However, the total motility of unwashed sperm samples extended with Bioxcell® had a higher percentage than other sperm sample groups. The percentage of spermatozoa VCL of washed semen samples extended with Triladyl® was significantly lower than the percentage of spermatozoa VCL of other treatment groups.
However, the lowest percentage of viable and normal spermatozoa was observed in the equilibrated sperm samples that were prolonged with Triladyl®. There was no significant difference (P > 0.05) in spermatozoa, abnormalities of bent midsection and coiled tail in unwashed and washed sperm samples extended with Bioxcell® and Triladyl®. However, the percentages of sperm head abnormalities in unwashed sperm samples were numerically lower than raw sperm samples, although there was no significant difference (P > 0.05).
There was no significant difference (P > 0.05) in the percentage of spermatozoa with normal acrosome in unwashed and washed sperm samples extended with Bioxcell® and Triladyl®. There was also a decrease in the percentage of normal chromatin spermatozoa from washed sperm samples extended with Triladyl®, compared to the other treatment groups. The percentage of spermatozoa with high mitochondrial membrane potential in unwashed and washed sperm samples extended with Bioxcell® was significantly lower (P < 0.05) than the unwashed and washed sperm samples extended with Triladyl®.
The effect of extenders and removal of seminal plasma on semen of South African
Effect of fillers and removal of seminal plasma on semen of South African indigenous goats after freezing and thawing. The effect of seminal plasma removal, egg yolk level and season on the freezeability of canary (Capra hircus) sperm. Effect of egg yolk and removal of seminal plasma fluid on semen cryopreservation in Norduz goats.
Effect of different degrees of dilution of frozen Angora semen with Bioxcell® extenders on post-thaw quality. Effect of sodium dodecyl sulfate (SDS) on viability and fertility of Damascus goat spermatozoa. Repeatability and effect of season on semen characteristics and computer-assisted semen analysis in the stallion.
Effect of three different extenders in slow freezing protocol on the quality of canine semen after thawing. Effect of seminal plasma removal, washing solutions and centrifugation regimes on cryopreservation of Boer goat sperm. Effect of extender and equilibration time on post-thaw motility and chromatin structure of buffalo bull (bubalus bubalis) spermatozoa.
Effect of different pre-storage equilibration periods on post-molting sperm motility in Nguni and Boran bulls. Comparison of the cryopreservation effect of different levels of egg yolk omega-3 in citrate extender on goat sperm quality.
