Effect of Heterologous Seminal Plasma and Semen Extenders on Motility of Frozen–thawed Ram Spermatozoa

INTRODUCTION Fresh ram semen may yield good fertility after cervical insemination, whereas frozen–thawed ram semen has to be deposited into the uterus for acceptable fertility. The need for uterine insemina-tion restricts the use of frozen–thawed ram semen because there is no method that is easy, repeatable, non-invasive and that does not require expensive equipment to achieve intrauterine insemina-tion. The addition of ram seminal plasma to frozen–thawed ram spermatozoa holds promise as it improves the fertility achieved with cervical insemination to acceptable levels 11. Ram semen contains relatively little plasma, which makes it an impractical additive where large numbers of ewes need to be inseminated with frozen–thawed semen. There is thus a


INTRODUCTION
Fresh ram semen may yield good fertility after cervical insemination, whereas frozen-thawed ram semen has to be deposited into the uterus for acceptable fertility.The need for uterine insemination restricts the use of frozen-thawed ram semen because there is no method that is easy, repeatable, non-invasive and that does not require expensive equipment to achieve intrauterine insemination.The addition of ram seminal plasma to frozen-thawed ram spermatozoa holds promise as it improves the fertility achieved with cervical insemination to acceptable levels 11 .Ram semen contains relatively little plasma, which makes it an impractical additive where large numbers of ewes need to be inseminated with frozen-thawed semen.There is thus a need to search for another fluid that would also yield acceptable fertility of frozen-thawed ram spermatozoa after cervical insemination.Like frozenthawed ram spermatozoa, frozen-thawed dog spermatozoa also result in high fertility after uterine insemination but poor fertility after vaginal insemination 1,7 .The addition of dog prostatic fluid to frozenthawed dog spermatozoa improves their fertility after vaginal insemination 12,13 .The ejaculate of the domestic dog contains a large amount of seminal plasma, largely derived from the prostate gland 2,3 .Adding bovine seminal plasma to frozenthawed African buffalo (Syncerus caffer) spermatozoa improves their ability to fertilise bovine oocytes in vitro, confirming that heterologous seminal plasma can have an effect on the fertility of spermatozoa 6 .
The motility of ram spermatozoa is considered essential for their migration through the cervix and from the central region of the cervical canal towards the cervical mucosa 9 where they are stored and released over time as required for fertilisation 10 .It is therefore possible that seminal plasma promotes the fertility of frozen-thawed ram spermatozoa deposited in the cervix by maintaining or promoting their motility.When searching for a fluid to replace ram seminal plasma as a post-thaw additive to frozenthawed ram spermatozoa prior to cervical insemination, its effect on the motility of frozen-thawed ram sperm should be compared to that of ram seminal plasma.
Heat-treated skim milk is an effective extender for fresh ram spermatozoa prior to cervical insemination 4 , resulting in high fertility, presumably due to its protein fraction, which may buffer against changes in pH 5 and partially protect spermatozoa during cooling for storage 15 .Adding dog prostatic fluid to frozenthawed dog spermatozoa not only improves their fertility after vaginal insemination 12,13 , but also their motility and longevity after thawing 14 .Adding protein-free TALP to frozen-thawed dog spermatozoa, however, maintains their post-thaw motility better than dog prostatic fluid 14 .
The aim of the current study is to compare the effects of bull seminal plasma, dog prostatic fluid, heat-treated skim milk and protein-free TALP on the motility of frozen-thawed ram spermatozoa to the effect of ram seminal plasma

Experimental animals
The semen donors were 2 Dorper, 2 Merino and 2 Dohne Merino rams, aged 2-4 years, kept at Faculty of Veterinary Science of the University of Pretoria in open-roofed pens with cement floors and fed lucerne hay and commercial sheep pellets, with water ad libitum.Semen was collected by means of an artificial vagina, using a non-oestrous ewe tied in a neck clamp as a mount.The ejaculates were collected during May, which coincides with the optimum breeding season for sheep in South Africa.3 straws individually allocated to each of 6 fluids that were added after thawing.In this way, the semen from 3 straws of each batch were separately diluted with the same type of fluid.The fluids were ram seminal plasma, bull seminal plasma, dog prostatic fluid, heat-treated skim milk, protein-free TALP and Triladyl with Equex STM paste (TrilEq).After thawing and dilution the semen was incubated at 37 °C for 6 h and the motility evaluated zero (t0), 2 (t2), 4 (t4) and 6 (t6) hours after thawing.The response variables were percentage progressively motile spermatozoa, percentage aberrantly motile spermatozoa and percentage immotile spermatozoa.

Collection, preparation and storage of fluid
TrilEq.Triladyl was prepared by adding 1 volume of Triladyl concentrate™ (Minitüb, Tiefenbach, Germany), which contains glycerol, tris, citric acid, fructose, tylosin, gentamicin, lincomycin and spectinomycin in unknown concentrations as this is a trade secret, to 3 volumes of deionised water and 1 volume of egg yolk.Finally, to each 100 m of Triladyl, 0.5 m of Equex STM paste (Nova Chemical Sales, Scituate, MA, USA) was added to give 100.5 m of TrilEq.TrilEq was used as freezing extender and as 1 of the fluids added to frozen-thawed semen.TrilEq was prepared in advance and stored at -70 °C until shortly before use.
Seminal plasma from rams and bulls and prostatic fluid from dogs.An ejaculate was collected from each of the 6 rams, pooled and centrifuged at 3500 g for 30 minutes and the supernatant filtered through a 0.22 µm filter.This process was repeated and the supernatants pooled until a sufficient quantity was available to yield 20 aliquots of 0.85 m of the sperm free, filtered seminal plasma that were transferred into Cryo-tubes (NUNC, Denmark) and stored at -70 °C.
One ejaculate from each of 8 bulls (2 Holsteins, 2 Ayrshires, 2 Tulis and 2 Brahmans) belonging to the Taurus Artificial Insemination Centre was collected by means of an artificial vagina and treated in the same manner as described for rams to yield 20 aliquots of seminal plasma.
The post-sperm fraction of 1 ejaculate from each of 6 dogs was collected by means of digital massage and then processed in the same manner as described for seminal plasma of rams and bulls to yield 20 aliquots of prostatic fluid.
Heat-treated skim milk.Ultra high temperature (UHT) skim milk (Clover South Africa (Pty) Ltd) contained 4.9 g carbohydrates, 3.4 g protein, 0.4 g milk fat and 123 mg calcium per litre.

Freezing of semen
One ejaculate was collected from each ram on each of 3 consecutive days and discarded, subsequent to which each ram rested for 3 days before another 3 ejaculates were collected from each, with a 60-minute interval between ejaculates.These last 3 ejaculates from each ram were extended, cooled to 5 °C, pooled and frozen as 1 batch per ram, as described below.Immediately after collection of an ejaculate destined for freezing, a small drop of it was suspended from a glass slide kept at 37 °C and examined under a microscope at ×40 magnification to confirm that the semen had strong wave movement (at least 4 on a scale from 0 to 5).Each ejaculate with adequate wave movement was immediately extended with 2 volumes of TrilEq at 30 °C in a 15-m tissue culture tube that was placed in a 50 m water jacket at 25 °C.This was placed in a beaker with 250 m water at 15 °C, which was transferred to a cool room at 5 °C.The 2nd and 3rd ejaculates from the ram were processed in the same way as the 1st and 150 minutes after the 3rd was placed in the cool room they were pooled in a 50 m tissue culture tube.The concentration of spermatozoa in the pool was determined by means of a haemacytometer and the pool further extended with TrilEq at 5 °C to a final concentration of 200 million per m .Two hours after the 3 ejaculates were pooled the suspension of spermatozoa was inverted repeatedly to ensure thorough mixing.Fifty 0.25 m French straws, marked with the name of the ram, were filled and sealed with PVC powder.The straws were frozen by suspending them horizontally 5 mm apart and 4 cm above liquid nitrogen in stable nitrogen in a Styrofoam box for 20 minutes before plunging them into the liquid nitrogen.The straws were then stored in a liquid nitrogen container at -196 °C.

Evaluation of sperm motility
In order to determine the sequence in which ejaculates and fluids had to be thawed, a random number was allocated to each ram-by-fluid-by-straw combination.The ram-by-fluid-by-straw combinations were then sorted in ascending order according to the random numbers, after which the straws were thawed, diluted, incubated and evaluated in the order of ascending random numbers.
One person thawed all straws and prepared all slides for the assessment of motility, using glassware kept at 37 °C, and another person performed all the assessments.Ten minutes before a straw with semen was thawed, the fluid destined for it was thawed and warmed to 37 °C.As soon as a straw with semen had thawed, its contents were transferred to a 4.5 m centrifugation tube and diluted with 0.85 m of the appropriate fluid.The person then placed a drop of the diluted semen onto the centre of a cover slip that was then inverted and placed on a glass slide labelled with the random number.This person then handed the slide over to the other who placed the slide on the warm stage (37 °C) of a phase-contrast microscope at ×200 magnification in order to assess the motility.The 2nd person estimated the percentages of progressively motile, aberrantly motile and immotile spermatozoa in 10 neighbouring fields along the equator of the coverslip, starting at the edge and ending at the centre 14 .From the results obtained average values were calculated.The individual sperm motility is expressed in terms of percentages of progressively motile, aberrantly motile and immotile spermatozoa.
The straw with the next higher random number was thawed and diluted 10 minutes after the previous one, and so on until all straws were evaluated.
The tubes containing the extended semen were capped and placed in a water bath at 37 °C until after the last evaluation at t6.

Statistical analysis
The statistical software package NCSS (www.ncss.com,Kaysville, UT USA) was used for all statistical analyses.Breed was initially included in the model, with ram nested in breed, but, because breed had no significant effect (P > 0.05) and breed was of no particular concern with respect to the aim of the study, it was removed prior to the final analysis.
A repeated measures ANOVA was used to test for differences among the postthaw motility with the rams as subjects and fluid and time as factors causing within-subjects variation.The interaction between fluid and time was included in the model, whereas the subject interactions were combined with error.Bonferroni's test was used to compare the mean of each fluid to that of ram seminal plasma, which served as control 8 .For all comparisons the level of significance was set to P < 0.05.

Fresh semen quality
Although there was a large variation in sperm concentration among rams, each ejaculate had good motility (mass motility score 5, percentage progressively motile spermatozoa exceeding 70 % and percentage aberrantly motile spermatozoa of 1 % or lower).

Motility after thawing and dilution
Ram, fluid and time significantly affected the percentage progressively motile spermatozoa, the percentage aberrantly motile spermatozoa and the percentage immotile spermatozoa (P < 0.001).Table 1 shows the mean percentages of progressively motile, aberrantly motile, and immotile spermatozoa for each fluid at different times of incubation.Ram seminal plasma resulted in a higher percentage progressively motile spermatozoa than bull seminal plasma and a lower percentage than heat-treated skim milk (P < 0.05), and a similar percentage to the other fluids (P > 0.05, Table 1).Ram seminal plasma resulted in a lower percentage aberrantly motile spermatozoa than protein-free TALP (P < 0.05), and a similar percentage to the other fluids.For all times combined, ram seminal plasma resulted in similar percentage immotile spermatozoa to dog prostatic fluid, which was lower than the percentage for bull seminal plasma, and higher than the percentages for other fluids (P < 0.05).Heat-treated skim milk resulted in a higher percentage progressively motile spermatozoa and a lower percentage immotile spermatozoa than all other fluids (P < 0.05).
The effect of ram seminal plasma and dog prostatic fluid on motility was strikingly similar at each time after the onset of incubation, as well as over all times combined (Table 1).
Fluid interacted with time with respect to percentage progressively motile spermatozoa, the percentage aberrantly motile spermatozoa and the percentage immotile spermatozoa (P < 0.001).
The interaction between fluid and time with respect to percentage progressively motile spermatozoa was mainly because the percentage progressively motile spermatozoa in spermatozoa samples diluted with bull seminal plasma declined more steeply during the 1st 2 hours of incubation than it did for spermatozoa diluted in other fluids.Fluid interacted with time with respect to the percentage aberrantly motile spermatozoa mainly because, for protein-free TALP, the percentage spermatozoa with aberrant motility increased by about 15 % during the 1st 2 hours of incubation and remained at a similar level during the following 4 hours, while no such rise occurred with the other fluids.The interaction between fluid and time with respect to percentage immotile spermatozoa was mainly because, for bull seminal plasma, the percentage immotile spermatozoa increased more steeply during the 1st 2 hours of incubation than for the other fluids.

DISCUSSION
Although ram seminal plasma improves the fertility of frozen-thawed ram spermatozoa deposited into the cervix 11 and heat-treated skim milk does not, ram seminal plasma does not preserve the in vitro motility of such spermatozoa as well as heat-treated skim milk.This observation suggests that ram seminal plasma exerts its beneficial effect on the fertility of frozen-thawed ram spermatozoa deposited into the cervix via a mechanism other than exceptional preservation of progressive motility.
Assuming that ram seminal plasma preserves progressive motility of frozenthawed spermatozoa that were deposited into the cervix sufficiently well to result in acceptable fertility, the results of the current study suggest that dog prostatic fluid and protein-free TALP, both of which preserved progressive motility as well as ram seminal plasma, should also adequately preserve their progressive motility.In the dog, however, vaginally inseminated frozen-thawed spermato-Table 1: Mean (± least squares error) for the percentages progressively motile spermatozoa, aberrantly motile spermatozoa (in brackets) and immotile spermatozoa (in square brackets) for frozen-thawed ram semen diluted and incubated in various fluids after thawing (n = 18 for each fluid by time combination and 72 for each fluid).

Post-thaw diluent
All times combined 0 2 4 6 zoa resulted in higher fertility after the addition of dog prostatic fluid than protein-free TALP 13 .The observation that seminal plasma of the domestic bull improves the ability of African buffalo spermatozoa to fertilise bovine oocytes in vitro 6 suggests that it is reasonable to suspect that effects of seminal plasma on fertility need not be restricted to an autologous effect.Sheep and dogs share certain characteristics with respect to the utility of frozenthawed spermatozoa in the 2 species: fertility resulting from deposition of frozen-thawed spermatozoa is good after deposition in the uterus, but poor after deposition at the external cervical opening, and fertility after deposition at the latter site is better if seminal plasma is added to the frozen-thawed spermatozoa prior to insemination.
Further research is required to compare the effects of ram seminal plasma and dog prostatic fluid on the fertility of frozenthawed ram spermatozoa deposited into the cervix.

REFERENCES
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