SEMEN EVALUATION METHODS IN CATTLE

Dr. Yahya Dahmani Magapor R&D Department

Yahya Dahmani, a member of the MAGAPOR R&D Dept and a specialist in the reproduction in the bovine species explains the methods used to assess and inspect bull semen in this article below.

Introduction Artificial insemination (AI) has proven to be the most effective tool for genetic improvement of animals of zootechnical importance, especially in the cattle industry. The inspection and handling of semen is considered a key and essential step for assessing fertility and the successful use of semen. Knowledge of fertility in bulls is an objective of great importance for the production of bovine semen, which is achieved by good analysis of the semen, among other assessments. To design an ideal bovine semen analysis to properly assess and predict the fertility of an ejaculate is the goal of many specialists in the reproduction field. The ideal semen analysis would be simple and effective, allowing the breeding capacity of a particular ejaculate to be predicted. A fertile ejaculate must meet certain semen parameter quality standards, such as: progressive motility, normal morphology, active energy metabolism, structural integrity and functionality of the membrane, penetration capacity and optimum transfer of genetic material. All current work on semen analysis seeks to identify some kinetic, morphological or biochemical parameters indicating the status of the sperm cell at any given time, while at the same time correlating with fertility and ejaculate quality. In routine production, the test should be accurate, simple, fast and economical.

Collecting bovine semen The semen extraction area must be as close as possible to the semen analysis laboratory (and not more than 30 metres). Semen is collected by several methods, the most common is the use of an artificial vagina. After the bull has mounted, the tube of semen is recovered from the artificial vagina and kept in a water bath at a

SEMEN EVALUATION METHODS IN CATTLE

temperature of 32-35C. This prevents temperature changes which can affect the quality of semen, which is then analysed.

After collection, semen analysis and assessment is divided into two parts: 1. General examination 2. Examination with a microscope.

Semen analysis 1. General examination The general examination consists of directly observing and examining the ejaculate in the tube to rule out any defect due to volume, colour, odour or dirt. Volume: This is a parameter that depends on the function of the semen vesicle and sex glands, plus other factors such as age, species, training etc, and varies from 1 to 8 mL. Most bulls provide about 6 mL of ejaculate. Colour: This depends on the amount of spermatozoa. When the semen is of good quality, it has a milky or creamy white colour and is of poor quality when its colour is similar to watered milk. If the ejaculate is yellow, it may be contaminated with urine and, if pink, there may be the presence of blood. Odour: In good condition, semen has a smell similar to fresh milk. The smell of urine indicates that the semen is contaminated with it. If the smell is unpleasant, some kind of disease is suspected in the testicles or elsewhere in the reproductive tract. Appearance: Its opacity, which depends on the concentration of spermatozoa, is assessed. pH: is determined using a paper strip between the range 6.4 to 6.9. Values above 6.9 are indicative of low semen quality.

2. Microscopic examination Microscopic examination is done under a bright field microscope to analyse the masal and individual motility of the spermatozoa, the morphology and concentration.

SEMEN EVALUATION METHODS IN CATTLE

Masal motility: is determined by placing a drop of semen on a slide, and then observing it under a bright field microscope with little magnification. The presence of waves and eddies throughout the whole drop is evaluated and is given a classification from 0 to +++, as below: Very good: Lots of dark waves moving rapidly (+++). Good: Less dark waves than the previous are observed, with moderate movement (++). Normal: Clear waves with very slight movement (+). Poor: No waves, and the spermatozoa are immobile (0).

Individual motility: This parameter can be analysed more objectively with automated systems such as the CASA system. However, it is also determined by bright field microscope examination by placing the slide on a drop of semen diluted with saline or sodium citrate (0.9%). A coverslip is then placed over it and observation performed under a bright field microscope with maximum magnification. The semen is classified according to the type of movement of the individual sperm, as follows: Very good: Equal to or greater than 70% progressive individual motility. Good: 50-69% individual motility. Normal: 30-49% individual motility. Poor: Less than or equal to 29% individual motility.

Sperm viability, acrosome and morphological abnormality is determined by microscopic observation of a smear of semen subjected to special staining fluids, usually eosin-nigrosin. The method involves placing a drop of approximately 10 microlitres of pure semen on a prepared slide, (cleaned and degreased at a temperature of 36-37 C on the hot plate). It is mixed gently with a drop of eosinnigrosin, which must be at the same temperature as the semen, using the pipette tip. Another slide, prepared as above, is supported on the edge of the drop until the liquid begins to spread over the slide by capillary action. It is then spread evenly, firmly and softly. The assessment of sperm viability, acrosomic and morphological abnormality is performed under a bright field microscope at x1000 magnification. A total of 200 sperms are counted and the percentage of non-viable cells, abnormal acrosomes and malformations of the sperm (coloured) are calculated. Another way to assess the acrosomic abnormality is by fixing with 0.2% glutaraldehyde and observing with a phase-contrast microscope. For morphology, Rose Bengal can also be used, and the bright pink sperms counted. Non-viable sperms are stained, and if its acrosome is present, a more intense violet can be seen. If this is absent, the

SEMEN EVALUATION METHODS IN CATTLE

head of the sperm is of a more homogeneous colour, or the upper third is lighter in colour.

Bovine sperm stained with eosin-nigrosin. Right: dead spermatozoa (red). Left: Living spermatozoa. The minimum value is 70% normal acrosomes. The abnormalities observed are classified as primary or secondary. This classification is most widely used in the literature, but not the most accurate. Primary malformations are, by definition, those that originate within the testes during spermatogenesis and secondary malformations are those that originate within the epididymis. At present, it is perhaps more appropriate to identify each of these abnormalities according to its location within the structure of the spermatozoon and the relationship of each with fertility. There is much still to be studied in this field. In general, the maximum number of head abnormalities allowed in the ejaculate is between 15 and 20%. Acrosome and tail abnormalities are acceptable up to 25%. In no case should less than 70% of normal spermatozoa in the ejaculate of a breeding bull be accepted.

Concentration: This is number of spermatozoa per mL, and is calculated by counting the sperm, with respect to the dilution and volume, in a counting chamber (Burcker or similar) under a bright field microscope. Another way to determine this semen parameter is examination with an automatic system, such as CASA, or the use of a spectrophotometer. The characteristic values of bovine semen for normal fertility are: Progressive motility: >50% Concentration: >500 million/mL Sperm vitality: >50% Abnormal heads: <20% (it is normally 8-12%) Proximal cytoplasmic droplets: <4% Distal cytoplasmic droplets <4% Abnormal middle piece: <15%, Double tails <4% Crooked tails <3%.

SEMEN EVALUATION METHODS IN CATTLE

3. Semen processing The purpose of semen processing is to keep the sperm fertile for as long as possible. Semen processing involves the following steps: dilution, stabilisation at 5C, filling in straws and freezing.

Dilution: Once the sperm concentration is determined, the amount of diluent or extender that can be added to the semen and the number of straws that can be made are calculated, based on the following formulas:

Vol. Dil = NP x 0.25 - V NP = V x MI x C (mL) / N of spermatozoa per straw Where: Vol. Dil: Volume of diluent NP: Number of straws 0.25: Straw volume in mL V = Semen volume in mL MI = Percentage individual motility C = Spermatozoa concentration in mL The number of spermatozoa per straw (dose) is usually 30 million.

Preservation at 5C: after calculating the dilution and the number of straws, the diluted semen is cooled from 25C to 5C (at a rate of 0.2-0.3C per minute), then stabilised at 5C for at least 2 hours before filling the straws.

Filling the straws: the straws are filled at 4C manually or automatically (with filling machines). The filling can be done at a laboratory temperature of 24C before cooling and stabilising at 4C. Once completed, the straws are sealed with polyvinyl material and frozen.

Freezing: After filling and sealing the straws and allowing to stabilise, the sperm is frozen. This process can be performed using liquid nitrogen or a biofreezer. The freezing temperature is typically -120C, and the typical freezing ramp is as follows: From 5 to -6C, 4C/min From -6C to -25C, 50C/min

SEMEN EVALUATION METHODS IN CATTLE

From -25C to -10C, 50C/min From -10C to -120C, 80C/min The straws are then immediately placed into liquid nitrogen to reach a final temperature of -196C.

4. Evaluation of frozen semen Post-thawing evaluation is done to ascertain whether the semen is able to overcome the freeze-thawing process. Factors that may affect the evaluation are due to the sperm itself or its handling (lack of liquid N2 in the storage container, handling of straws, etc). The thawing of straws is performed in water at 37C for 30-50s. After this, one end of the straw is cut and put in a tube inside a water bath, the other end is then cut to empty the contents. The evaluation of the semen dose consists of the following steps:

a) Motility examination: The percentage progressive motility and vigour are determined immediately after thawing the semen and after 2 hours of incubation. Various methods can be used to determine the motility. Automated systems, such as CASA, are the most objective. With experience, bright field microscopic examination without counting cells provide quick estimates. A drop of semen is spread thinly and evenly between a warm slide and coverslip at a magnification of 1000 to evaluate the percentage of spermatozoa with progressive motility, then the rate of progression (vigour) is assessed on the following scale: 0 = No movement. 1 = Slight ripple or vibration at the tail, without progression. 2 = Slow progression, including stop and start movements. 3 = Continuous progressive movement at a moderate speed. 4 = Progressive and rapid movement. 5 = Very fast progressive movement, with the cells being difficult to follow visually. Semen of good quality, which has been recently thawed, usually has 40-50% of spermatozoa with progressive motility with a vigour of 3-4. After 2 hours of incubation, these values usually decrease by 10-15% and 1 degree, respectively. The minimum standards required for motility are defined by the Department of Medicine and Bovine Theriogenology at the University of Saskatchewan, Canada corresponding to those of ISO 9002, which are: 0 hrs= 25% of spermatozoa with progressive motility. Vigour 3. 2 hrs= 15% of spermatozoa with progressive motility. Vigour 2. b) Percentage of intact acrosomes: Determining the percentage of intact acrosomes is a morphological method for measuring the viability after thawing, and is related to fertility. Determining the viability and potential fertility of frozen semen is a valuable addition to motility. To examine the acrosome, sperm movement must

SEMEN EVALUATION METHODS IN CATTLE

be stopped. This is accomplished by mixing the semen with 0.2% buffered glutaraldehyde, which fixes the membrane and prevents its further deterioration. The preparation can be done on the slide, by placing a small drop of semen next to a droplet of glutaraldehyde, mixing well and placing a coverslip over it. Two hundred cells are examined at a magnification of 1000, immediately after thawing the semen and after 2 hours of incubation. The minimum satisfactory values are: 0 hrs= 50% of acrosomes intact. 2 hrs= 35% of acrosomes intact. c) Viability and morphology: The determination of these parameters is done using the same methodology as for fresh semen, with minimum values for viable and normal spermatozoa of 40% and 70%, respectively. d) Concentration: The total number of spermatozoa per straw (of 0.25 or 0.5 mL) varies from 20 to 30 million, of which there must be at least 10 million motile cells after thawing. To evaluate the concentration, the contents of a straw are diluted 1:200 in saline formalin and counting is performed in a counting chamber. The total number of motile spermatozoa per dose is then calculated. e) Cell Endosmosis test (HOST, hypoosmotic swelling test): This evaluates the functionality of the sperm membrane by testing its ability to respond to osmotic changes. This ability is related to the functional capacity of the membrane (functioning of ion channels, Na+/H+ exchange, etc). After being subjected to hypoosmotic media, these cells absorb water providing the sperm membrane is not damaged. If the cells have their membranes intact, there will be an influx of water from the media which is incorporated in the tail, which will be swollen and coiled. The semen is incubated for 10-15 minutes at 37C in a hypoosmotic fructose and sodium citrate solution (75 mOsm/kg). Observations after 0, 1 and 2 hours are performed on the slide under a bright field microscope at 1000 magnification, and the percentage of live spermatozoa determined. A total of 200 spermatozoa are counted and the percentage of cells that respond to the osmotic shock by coiling their tail is calculated. At least 40% of the sperm must react. f) Post-thawing quality control The quality of the thawed semen dose should be as follows: Thawing temperature: 37C Individual Progressive Motility: > 40% and Vigour: 3-4 ( on a scale of 0-5) Normal sperm morphology: >70% Acrosome integrity: > 60% HOST Test: >40% of spermatozoa reacting. Minimum number of viable sperm: (40%)