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Quality of Ram Spermatozoa Separated with Modified Swim Up Method

A Hossain
Department of Surgery and Obstetrics, Faculty of Veterinary Science, Bangladesh Agricultural University, Mymensingh -2202, Bangladesh

M M Islam
Department of Surgery and Obstetrics, Faculty of Veterinary Science, Bangladesh Agricultural University, Mymensingh -2202, Bangladesh

F Naznin
Department of Surgery and Obstetrics, Faculty of Veterinary Science, Bangladesh Agricultural University, Mymensingh -2202, Bangladesh

R N Ferdousi
Department of Surgery and Obstetrics, Faculty of Veterinary Science, Bangladesh Agricultural University, Mymensingh -2202, Bangladesh

F Y Bari
Department of Surgery and Obstetrics, Faculty of Veterinary Science, Bangladesh Agricultural University, Mymensingh -2202, Bangladesh

N S Juyena
Department of Surgery and Obstetrics, Faculty of Veterinary Science, Bangladesh Agricultural University, Mymensingh -2202, Bangladesh

Abstract/ Executive Summary:

Semen was collected from four rams, using artificial vagina and viability%, motility% and plasma membrane integrity% were measured. Fresh ejaculates (n = 32) were separated by modified swim-up separation using modified human tubal fluid medium. Four fractions of supernatant were collected at 15-minute intervals. The mean volume, mass activity, concentration, motility%, viability%, normal morphology and membrane integrity% (HOST +ve) of fresh semen were 1.0 ± 0.14, 4.1 ± 0.1 × 109 spermatozoa/ ml, 85.0 ± 1.3, 89.4 ± 1.0, 85.5 ± 0.7, 84.7 ± 0.5 respectively. There was no significant (P>0.05) difference in fresh semen quality parameters between ra ms. The motility%, viability% and HOST +ve % of first, second, third and fourth fractions were 53.4 ± 0.5, 68.2 ± 0.3, 74.8 ± 0.3 and 65.5 ± 0.4; 55.5 ± 0.4, 66.2 ± 0.4, 74.5 ± 0.3 and 73.6 ± 0.3 and 66.7 ± 0.5, 66.8 ± 0.5, 65.2 ± 0.4 and 74.7 ± 0.5 respectively. The motility%, viability% and membrane integrity% of separated semen samples differed significantly (P<0.05) between four fractions. The mean motility% and viability% were significantly higher (P<0.05) in third fraction (74.8 ± 0.3%), whereas the mean HOST +ve% was significantly higher (P<0.05) in fourth fraction (74.7 ± 0.5). All quality parameters of separated spermatozoa were significantly (P<0.05) lower than that of fresh semen. The pregnancy rates were higher with fresh semen (71%) in comparison to that of separated sample (57%).

Keywords: Ram, Semen, Pregnancy, Spermatozoa, Indigenus

Location: Research Animal Farm, Department of Surgery and Obstetrics, Faculty of Veterinary Science, BAU

Start Date: 01-07-2016

End Date: 31-12-2016

Program Area: Animal Health and Management

Commodity: Sheep

Objectives:

i. To evaluate quality of separated spermatozoa of indigenous rams; and

ii. To compare quality of normal and separated spermatozoa.

Methodology:

The research was conducted at the Research Animal Farm, Department of Surgery and Obstetrics, Faculty of Veterinary Science, Bangladesh Agricultural University, Mymensingh -2202, from July to December 2016. Selection and management of rams: Four healthy indigenous rams, from 1.5 to 3 years old, were selected as semen donors. Body weight and scrotal circumference of these ram varied from 15 to 20 kg and 18 to 25 cm, respectively. All rams were apparently healthy. They were housed in a covered shelter with an open -air run and allowed 6 to 8 hours natural grazing. Routine vaccination and deworming programs were conducted. Each ram was fed approximately 0.5 to 1.0 kg concentrate daily. Experimental design: Semen collection and evaluation: Semen was collected by artificial vagina (AV) using a homosexual teaser twice a week. Before semen collection warm water (40°C to 45°C) and air were injected into the AV. The rubber part of the AV was lubricated by sperm-friendly lubricant. Semen in vials was kept in a water bath (37^oC) as early as possible. Eight ejaculates were collected from each ram. Immediate after collection, volume of semen was recorded. Colour of the semen was scored by naked eye 1–5. Only samples scored 3 or more were accepted for processing. Mass motility was scored from 1–5 using phase-contrast microscopes. A small drop of fresh semen was placed on pre -warmed (37°C) grease -free slide and observed under microscope at 10 ´ without cover slip, and mass activity (0–5) score was recorded. The concentration of spermatozoa (billion/ mL) was determined by using haemocytometer. Semen samples were diluted with distilled water (1 : 400) to kill the spermatozoa. A drop of diluted semen was placed on the counting chamber from a pipette, and spermatozoa were allowed to settle for 5–6 minutes before placing the haemocytometer on the stage of the microscope. The concentration of spermatozoa per ml of semen was calculated by multiplying the total number of spermatozoa in 5 large squares by 2 × 10 7. Percentage of progressively motile spermatozoa was measured by phase -contrast microscope: 80% or more was considered acceptable. For sperm motility, a small drop (5 µL) of diluted semen was placed on a clean pre -warmed glass slide and covered with a cover slip. Motility was determined by eye under 40x magnification and expressed as percentage. The mo rphology of spermatozoa is used as an important criterion in the evaluation of semen. Spermatozoa having no abnormalities with respect to acrosome, midpiece and tail were considered as normal spermatozoa. Air dried smear of semen was stained with Farely stain as per as manufacturer guideline. After drying, the stained smears were examined under microscope (x100) using oil immersion . Cell viability assessment: 5 µL Propidium iodide (PI) of semen sample was diluted with 1950 µl of saline extender (2.9% Na -citrate) and 20 µL of 6-CFDA, 20 µL of PI and 20 µL of formaldehyde were added. Stained sample was then incubated at 37^oC for 15 min in dark and evaluated using fluorescence microscope. Viable sperm% (CF + PI-, green) and dead% (CF-PI+, red) was recorded. Sperm plasma membrane functional integrity: The functional integrity of spermatozoa was evaluated by using hypo-osmotic swelling test (HOST). Osmolar solution (100 m Osm/ L) wi th sodium citrate and fructose was use. A 1.0mL of hypo -osmotic solution was mixed with 0.1ml of undiluted semen and incubated at 37°C for 1 hour. After incubation, 200 spermatozoa were examined under phase contrast microscope (400x) and percentage of spermatozoa positive to HOST (with bent tail) was recorded. Swim-up separation: Semen (0.25 mL) was mixed with mHTF (1.0 mL). The supernatant was collected and evaluated 4 times. Test tube was placed in a dry bath for 60 min for incubation. Motile spermatozoa were collected after swimming up after 15, 30, 45 and 60 minutes. Sperm from all supernatant samples were evaluated. Sperm of second fraction of supernatant was used for artificial insemination. Oestrous synchronization: Oestrus was induced in the ewes by intramuscular administration of two injections of PGF2∞ @ 100 µg (0.5 mL) per ewe. The first injection was given ignoring the stage of the oestrous cycle. After 9 days the doses were repeated to all ewes regardless of oestrus. Teaser ram was used to detect the onset of oestrus. Process of cervical AI: Basic equipment consisted of a speculum with a built -in light source, and a pipette connected to a 1 mL syringe. The vulva of the ewe was wiped with cotton wool. The speculum with lubricant was carefully inserted into the vagina to a depth of 10-13 cm. The cervix opening was located. The plunger of the syringe was withdrawn to 0.2 mL to have some air behind the semen. The appropriate amount of semen was drawn into the pipette from the semen collection tube in a water bath at 30°C–34°C. The inseminator attempted to introduce the pipette into the cervix without using force. Semen was deposited into the cervix. The speculum was withdrawn before the pipette to prevent backflow of the semen. Pregnancy diagnosis: Ewes were monitored for non-return to oestrus by the aid of a vasectomized teaser ram at 15–17 days following insemination. Pregnancy was confirmed using MUIV ultrasonography (Korea) with transducer frequency 5 MHz 45–55 days after insemination. Statistical analysis: All data were entered in Excel program. Values relating to semen evaluation were expressed as mean ± Standard Error (SE). Two -way analysis of variance (ANOVA) was done to find out significant differences in semen parameters between rams. Pearson’ correlation test was performed for the quality parameters of separated spermatozoa. All the statistical analyses were done using SPSS 17.0. Differences were regarded as significant when P was less than 0.05 (P<0.05).

Source of Information: The Bangladesh Veterinarian (2016) 33(2) : 62 – 70

Article Link (Online Journal):

Funding Source:

1. University Grant Commission

Budget:

Total Budget (Tk.) :

Achievement/Findings:

The study concludes that there was no significant (P> 0.05) difference in fresh semen quality parameters between the four rams tested. The motility%, viability% and plasma membrane integrity varied significantly (P<0.05) between fractions and between rams. Pearson’s correlation showed a strong positive (P<0.0 01) relation between viability% and motility%.

Knowledge Management: Journal