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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 32-35ºC. Values above 6. training etc.9 are indicative of low semen quality. species. Volume: This is a parameter that depends on the function of the semen vesicle and sex glands. it may be contaminated with urine and. is assessed. if pink. General examination 2. Examination with a microscope. odour or dirt. After collection. semen has a smell similar to fresh milk. it has a milky or creamy white colour and is of poor quality when its colour is similar to watered milk. plus other factors such as age. and varies from 1 to 8 mL. Most bulls provide about 6 mL of ejaculate. there may be the presence of blood. colour. which depends on the concentration of spermatozoa. semen analysis and assessment is divided into two parts: 1. which is then analysed. Appearance: Its opacity. When the semen is of good quality. The smell of urine indicates that the semen is contaminated with it. Odour: In good condition. This prevents temperature changes which can affect the quality of semen. pH: is determined using a paper strip between the range 6. Microscopic examination Microscopic examination is done under a bright field microscope to analyse the masal and individual motility of the spermatozoa. Colour: This depends on the amount of spermatozoa. 2 . 2. General examination The general examination consists of directly observing and examining the ejaculate in the tube to rule out any defect due to volume. some kind of disease is suspected in the testicles or elsewhere in the reproductive tract.4 to 6.9. the morphology and concentration. If the smell is unpleasant. If the ejaculate is yellow. Semen analysis 1.
Another way to assess the acrosomic abnormality is by fixing with 0. is supported on the edge of the drop until the liquid begins to spread over the slide by capillary action. a more intense violet can be seen. A total of 200 sperms are counted and the percentage of non-viable cells. acrosome and morphological abnormality is determined by microscopic observation of a smear of semen subjected to special staining fluids. Normal: 30-49% individual motility. For morphology. Another slide. Rose Bengal can also be used. and then observing it under a bright field microscope with little magnification. The method involves placing a drop of approximately 10 microlitres of pure semen on a prepared slide. acrosomic and morphological abnormality is performed under a bright field microscope at x1000 magnification. The assessment of sperm viability. 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. Normal: Clear waves with very slight movement (+). Individual motility: This parameter can be analysed more objectively with automated systems such as the CASA system. as below: Very good: Lots of dark waves moving rapidly (+++). It is then spread evenly. A coverslip is then placed over it and observation performed under a bright field microscope with maximum magnification. Good: Less dark waves than the previous are observed. Good: 50-69% individual motility. However. the 3 . (cleaned and degreased at a temperature of 36-37 °C on the hot plate). as follows: Very good: Equal to or greater than 70% progressive individual motility. which must be at the same temperature as the semen. firmly and softly.SEMEN EVALUATION METHODS IN CATTLE Masal motility: is determined by placing a drop of semen on a slide. and the bright pink sperms counted. If this is absent.2% glutaraldehyde and observing with a phase-contrast microscope. Poor: Less than or equal to 29% individual motility. The semen is classified according to the type of movement of the individual sperm. Poor: No waves. It is mixed gently with a drop of eosinnigrosin. abnormal acrosomes and malformations of the sperm (coloured) are calculated. Non-viable sperms are stained. usually eosin-nigrosin.9%). Sperm viability. The presence of waves and eddies throughout the whole drop is evaluated and is given a classification from 0 to +++. with moderate movement (++). prepared as above. and if its acrosome is present. using the pipette tip. and the spermatozoa are immobile (0).
Primary malformations are. In general. those that originate within the testes during spermatogenesis and secondary malformations are those that originate within the epididymis. 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. with respect to the dilution and volume. Double tails <4% Crooked tails <3%. Concentration: This is number of spermatozoa per mL. 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%. and is calculated by counting the sperm. the maximum number of head abnormalities allowed in the ejaculate is between 15 and 20%. Left: Living spermatozoa.SEMEN EVALUATION METHODS IN CATTLE head of the sperm is of a more homogeneous colour. by definition. or the use of a spectrophotometer. This classification is most widely used in the literature. Acrosome and tail abnormalities are acceptable up to 25%. There is much still to be studied in this field. The abnormalities observed are classified as primary or secondary. Another way to determine this semen parameter is examination with an automatic system. At present. in a counting chamber (Burcker or similar) under a bright field microscope. In no case should less than 70% of normal spermatozoa in the ejaculate of a breeding bull be accepted. or the upper third is lighter in colour. 4 . but not the most accurate. Bovine sperm stained with eosin-nigrosin. such as CASA. The minimum value is 70% normal acrosomes. Right: dead spermatozoa (red).
Once completed. the sperm is frozen.V NP = V x MI x C (mL) / N° of spermatozoa per straw Where: Vol. filling in straws and freezing. The filling can be done at a laboratory temperature of 24°C before cooling and stabilising at 4°C.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. the amount of diluent or extender that can be added to the semen and the number of straws that can be made are calculated. Dil = NP x 0. Preservation at 5°C: after calculating the dilution and the number of straws. 4ºC/min From -6ºC to -25ºC.SEMEN EVALUATION METHODS IN CATTLE 3.3°C per minute). Filling the straws: the straws are filled at 4°C manually or automatically (with filling machines). Dilution: Once the sperm concentration is determined. Dil: Volume of diluent NP: Number of straws 0. 50ºC/min 5 . The freezing temperature is typically -120°C. the diluted semen is cooled from 25°C to 5ºC (at a rate of 0. and the typical freezing ramp is as follows: From 5 to -6ºC.25 . then stabilised at 5ºC for at least 2 hours before filling the straws. Freezing: After filling and sealing the straws and allowing to stabilise. stabilisation at 5°C. the straws are sealed with polyvinyl material and frozen.2-0. based on the following formulas: Vol. Semen processing The purpose of semen processing is to keep the sperm fertile for as long as possible. This process can be performed using liquid nitrogen or a biofreezer. Semen processing involves the following steps: dilution.
etc). 2 = Slow progression. then the rate of progression (vigour) is assessed on the following scale: 0 = No movement. respectively. 4 = Progressive and rapid movement. 4. 5 = Very fast progressive movement. 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. To examine the acrosome. Vigour 3. Factors that may affect the evaluation are due to the sperm itself or its handling (lack of liquid N2 in the storage container. 3 = Continuous progressive movement at a moderate speed. including stop and start movements. bright field microscopic examination without counting cells provide quick estimates. which are: 0 hrs= 25% of spermatozoa with progressive motility. sperm movement must 6 . With experience. The minimum standards required for motility are defined by the Department of Medicine and Bovine Theriogenology at the University of Saskatchewan. the other end is then cut to empty the contents. one end of the straw is cut and put in a tube inside a water bath. 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. Semen of good quality. which has been recently thawed. Various methods can be used to determine the motility. b) Percentage of intact acrosomes: Determining the percentage of intact acrosomes is a morphological method for measuring the viability after thawing. handling of straws. 80ºC/min The straws are then immediately placed into liquid nitrogen to reach a final temperature of -196°C. 2 hrs= 15% of spermatozoa with progressive motility. After 2 hours of incubation. Automated systems. are the most objective. these values usually decrease by 10-15% and 1 degree. without progression. Evaluation of frozen semen Post-thawing evaluation is done to ascertain whether the semen is able to overcome the freeze-thawing process. Vigour 2. such as CASA. Canada corresponding to those of ISO 9002. 1 = Slight ripple or vibration at the tail. The thawing of straws is performed in water at 37ºC for 30-50s. and is related to fertility. usually has 40-50% of spermatozoa with progressive motility with a vigour of 3-4. with the cells being difficult to follow visually.SEMEN EVALUATION METHODS IN CATTLE From -25ºC to -10ºC. After this. Determining the viability and potential fertility of frozen semen is a valuable addition to motility. 50ºC/min From -10ºC to -120ºC.
SEMEN EVALUATION METHODS IN CATTLE be stopped. by placing a small drop of semen next to a droplet of glutaraldehyde. which fixes the membrane and prevents its further deterioration. Observations after 0. The total number of motile spermatozoa per dose is then calculated. Minimum number of viable sperm: (40%) 7 . hypoosmotic swelling test): This evaluates the functionality of the sperm membrane by testing its ability to respond to osmotic changes.25 or 0. respectively. e) Cell Endosmosis test (HOST. The semen is incubated for 10-15 minutes at 37ºC in a hypoosmotic fructose and sodium citrate solution (75 mOsm/kg).2% buffered glutaraldehyde. After being subjected to hypoosmotic media. there will be an influx of water from the media which is incorporated in the tail. To evaluate the concentration. d) Concentration: The total number of spermatozoa per straw (of 0. The minimum satisfactory values are: 0 hrs= 50% of acrosomes intact. of which there must be at least 10 million motile cells after thawing. c) Viability and morphology: The determination of these parameters is done using the same methodology as for fresh semen. A total of 200 spermatozoa are counted and the percentage of cells that respond to the osmotic shock by coiling their tail is calculated. f) Post-thawing quality control The quality of the thawed semen dose should be as follows: Thawing temperature: 37°C 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. 1 and 2 hours are performed on the slide under a bright field microscope at 1000 magnification. Two hundred cells are examined at a magnification of 1000. which will be swollen and coiled. This is accomplished by mixing the semen with 0. with minimum values for viable and normal spermatozoa of 40% and 70%. the contents of a straw are diluted 1:200 in saline formalin and counting is performed in a counting chamber. At least 40% of the sperm must react. immediately after thawing the semen and after 2 hours of incubation. mixing well and placing a coverslip over it.5 mL) varies from 20 to 30 million. etc). and the percentage of live spermatozoa determined. If the cells have their membranes intact. This ability is related to the functional capacity of the membrane (functioning of ion channels. 2 hrs= 35% of acrosomes intact. Na+/H+ exchange. these cells absorb water providing the sperm membrane is not damaged. The preparation can be done on the slide.