Chapter 4 Factors Affecting The Function Of Sperm In Vitro Preparation Of Extender Extension Of Semen Principle Of Cryopreservation Steps In Cryopreservation Preservation Of Semen At Different Temperature@ FACTORS AFFECTING QUALITY AN QUANTITY OF SEMEN IN VIVO Age: Immature males produce poor quality semen. Bulls from 2 to 5 years of age produce larger volumes of ejaculate compared to bulls of less than 2 years of age and bulls older than 5 years. With advancement of age frequency of sperm abnormalities increases. Semen quality characteristics such as gel free volume, sperm concentration, total sperm number and sperm abnormalities are poorest in stallion under years of age and over 11 years of age.riod (season) Light appears to have minimal effects on semen quality in bulls. In ram, semen quality is best during periods of decreasing day | In horse, gel volume decreases to negligible amounts in the fall and winter with some decline in gel-free volume. Spermatozoal output during the winter months is about half of that during the breeding seasonTemperature spermatogenesis in most mammals (except elephant and whale) requires 6°C lower than body temperature. temperature 2to frost bite of scrotum and retention Elevated Body temperature, high fever, of testes within the abdominal cavity adversely affect seminal quality, depending on the duration and intensity of heat7) Nutrition Poor nutrition in general and protein deficiency causes deleterious effect on general health tryptophan, f vitamin (A, B & E), amino acids (arginine, lysine, ulting Deficiency o| yialanine and histidine) and minerals (Zn) affects the testes directly rest phen} into abnormal spermatogenesis Zine deficiency affects morphology and abnormalities in semen as it is associa with the attachment of the head to the tail. Zn - antioxidant properties. Prolonged dietary vitamin A deficiency impairs semen quality and semen production.DISEASES Varicocele, unilateral or bilateral cryptorchidism, epididymitis and spermatocele, seminal vesiculitis, genetic disorders like XXY syndrome, inter sex, hermaphroditism Dag defect, diadem defect, cork screw defect anc pseudodroplet defect - sub-fertility in bullsW Frequency of semen collections: *For liquid semen and frozen semen, collection is done thrice a week and twice a week, respectively. *Teasing and false mounts improve sperm concentration and semen volume. Frequent semen collection with poor sexual stimulation reduces semen quality. Management at the time of semen collection: “includes interval since last ejaculation, sexual Preparation, kind and size of semen collection equipment used proper handling of semen collection equipment.Pollution Chemicals causing environment pollution are known as endocrine disrupting compounds. Chemicals with anti-androgenic properties that have nin the environment have significant effects on the development and functioning of accessory sex glands and may be partly responsible for the decline in semen@ FACTORS AFFECTING THE FUNCTION OF SPERM IN VITRO + Temperature: The optimal temperature for the sperm motility is 37- 38°C. At higher temperatures sperm show unusually high motility and at 54-56°C sperm die at once. In contrast, when kept under 35°C the sperm motility gradually decreases. * Osmolarity: Semen and extender have similar osmolarity (285: mos/kg). When sperm is incubated in the medium with a lower osmolarity than this, the number of sperm having a bent tail increaseFACTORS AFFECTING THE FUNCTION OF S' ERM IN VITRO + Dilution: Excessive dilution (> 1: 1000) depresses the sperm motility mainly because of the dilution shock. Dilution of semen is not entirely straightforward, for mammalian spermatozoa placed in simple diluents exhibit an initial increase in motility, which is then rapidly followed by a loss of motility and increase in vital staining (Mann 1964).: in the acidic medium the motility of sperm is reduced, while in the basic medium it is enhanced. In the basic medium sperm has a vivid motility, but its viability is decreased. d radial The direct rays of the sun, ultraviolet and radial rays are detrimental to sperm. + Especially the radial rays damage DNA of sperm even at the level that has no effects on the sperm viability.® Gases: Oxygen promotes the motility and the metabolism of sperm, but the excessive oxygen reversibly inhibits the motility of sperm. ig detrimental to sperm. Carbon dioxide «Chemicals: such as caffeine and theophylline, acetylcholine and adrenaline enhance the motility of sperm even at low concentrations Vitamins A and B are effective for the maintenance of the motility. + Jons: The high concentration of potassium in the medium reduces the sperm motility and sodium neutralizes this effect. optimal ratio of K/Na is important for the maintenance of sperm motility.Semen of alpacas and llamas is highly viscous and mak ‘on, morphology and motili assessment of sperm concentrati difficult. + A comparatively high proportion of morphologic abnormaliti (up to 40%) are common. + Unlike the progressive motility of sperm seen in other domestic nants, only o: t vement is seen in the e,Properties of ideal men extend It provides volume to the semen with survivability of sperm Spermatozoa requires nutrition. So, it should nutrient (glucose) for aerobic and anaerobic metabolic processes. (6.8) it should have buffering capacity to maintain nearly neutral pH required for survival of sperm. It should maintain osmotic pressure as hypertonic and hypotonic solutions have adverse effects of sperm survival. It should have osmotic pressure of 285-300 milliosmoles which is equi nt to blood, semen and milk.lecithin on for protection against cold shock (e.g. s in egg yolk, phospholipids in milk and glycerol in deep freezing). Antibiotics to cover broad range of bacteria. The component of extender must be easily available and economicalPREPARATION OF EXTENDER Preparation of Tris buffer: The composition of Tris buffer is: 30.2849 16.750 Tris (Hydroxy methyl amino methane) Citricacid Fructose 10 to 12g Glass distilled water up to 1000mI Penicillin 10 lacks units/L Streptomycin tgittion room sterilized Buffer should be prepared in a separate prepa mum Standard Protocol (MSP) and all regularly by formalin as per Mini working areas should be sterilized with 70% alcohol. The buffer is autoclaved at psi for a period of 10 minutes The autoclaved buffer is cooled and after cooling the pH of buffe should be measured. The pH should be between 6.7 and 6.9 If the buffer is not used immediately it should be stored Tefrigerator at 4 to 8°CWw Preparation of egg yolk Purchase unfertilized fresh egg (not more than 4 to § days old). Wash, rinse, dry and store in a refrigerator until use. Wash the egg with warm water and wipe with 70% alcohol and allow drying. Crack and break the shell at the narrow end of the egg with the help of sterilized forceps. Make a hole in the egg shell by removing cracked shell. Pour the albumin in a beaker. Increase the hole size so that egg yolk can come out easily,@® Preparation of egg yolk Collect egg yolk gently on autoclaved filter paper in such a way that membrane should not rupture. Remove all white (albumin) by gently rolling the yolk on the filter paper. Puncture the yolk membrane with a sterilized object and collect yolk in sterilized graduated cylinder. The membrane of the yolk should remain on the filter paper and discarded. Properly mix egg yolk in the beaker with the help of sterile rod, especially when it has been obtained from more than one egg Glycerol is most widely used cryopreservative (7%) for citrate yolk and Tris egg yolk extenders; 10% in milk extenders.All steps of egg yolk preparation should be done in laminar flow.Preparation of Tris egg extender Composition of extender: Egg yolk 1 part or 20 ml 4 part or 80 ml Tris buffer 7% or 7 ml in 83 ml of above mixed egg yolk and Glycerol (viv) Tris buffer solution. The extender should be prepared fresh on the day of collection early in the morning or on the previous day in the evening. If prepared on the previous day, it may be noted that antibiotics should be added only in the morning prior to use.Preparation of Tris egg yolk citrate extender Glycerol can be warmed at 60°C temperature after measuring to increase its solubility or added to the buffer itself prior to sterilization/autoclaving on the previous day. The diluent is mixed gently and transferred to a conical flask. A sterilized magnet is put into the conical flask, and then conical flask is placed on a magnetic stirrer for a period of 30 minutes for homogenization.Commercial diluents: supplied by Minitub Germany and used with frozen e dilution semen. It consist of two fractions A and B for step wis of semen with addition of egg yolk, water and antibiotics. yi: supplied by Minitub Germany and used with frozen semen and is used as one step dilution medium and is similar t Biladyl. Boiciphos: supplied by IMV, L’Aigle, France. It is one step dilution medium containing soyabean extract replacing the egg yolk fraction.d milk ba’ AV, L’Aigle, France. It is skimme Laciphos: supplied by IM powder medium requiring addition of water. diluent for ok concentrated liquid semen Caprogen® concentrate: (18 to 240C) supplied by Liv storage at ambient temperatur Improvement, New Zealand. It is a diluent extensively used for liqu rage of semen with no drop in fertility over a 4-day period. Tris concentrate: Gibco BRL, manufactured by Holland G is most common diluent for freezing bovine semen used for liquid nW EXTENSION OF SEMEN itis necessary to double the number Up to 50% of sperm may die in the freezing process, if 10 million progressively motile sperm post-thaw are the goal, of sperm per dose; i.e., perm per dose would be 20 million progressively motile s the number of s| perm. Calculation of doses and dilution: Suppose one ejaculate has: Volume=5 ml, Spermconcentration= 1000 million perm! & Progressive motility Now calculate the volume of extender to be added and total number of mini stra ml) can be filled. Total number of sperm in this ejaculate = volume of the ejaculate x concentration =5x 1000 = 5000ively motile sperm in this ejaculate progressive motility) /100 Total number of progress! (Total number of sperm in this ejaculate x % of 15000 x 70)/100 = 3500 Total number of straws = Total number of progressive motile sperm in the ejaculate! 20 million progressive motile sperm = 3500/20 = 175. Total volume of extended semen (ml) = Total number of straws x Volume o: = 175 x 0,255 43.75 Volume of extender required (ml) = Total volume of extended semen — volume of ejaculateExtension or dilution of semen 38.75 ml extender will be gle Thus, in this 5 ml ejaculate, d 175 doses will be made from this sin added slowly an ejaculate. Nowadays photometer is utilized for this purpose. This instrument directly calculates the concentration of ejaculate, total number of straws and final volume of extended semen.ome of the but include: Phosphate buffer (Problem: head to head agglutination, ility) Citrate buffer (Most common) Tris (Tris-hydroxymethyl-amino-methane) TES (Tris-hydroxymethyl-methyI-: aminoethane sulphonic a TEST (Tris titra with TES)