Improving the quality of rooster semen frozen in straws by screening the glycerol concentration and freezing rate

ABSTRACT

• 1. This study examined different glycerol concentrations (GC) and freezing rates to improve the quality of rooster spermatozoa frozen in straws, and to determine the effect of varying GC on post-thawed spermatozoa quality, as evaluated by fertility and hatchability.

• 2.The experiment included two tests. In test 1, rooster semen straws containing 2, 4, 6, 8 and 11% glycerol were put in a rack (nine tiers with a 1 cm interval between every two tiers, 1 to 9 cm above liquid nitrogen (LN) source), and gradually frozen. The semen straws located in different tiers experienced different temperatures and freezing rates. The straws were then thawed and live sperm numbers determined. In test 2, rooster semen straws containing 2, 4, 6, 8 and 11% glycerol were put on optimal tiers (identified in test 1) for freezing, and stored at ?196°C. Hens were inseminated with the frozen semen (post-thawed and glycerol removed, about 4.0 × 10⁸ sperm per hen), and eggs incubated.

• 3. The numbers of live sperm in the 11% glycerol group was higher than that in 2, 4 or 6% glycerol group (P < 0.05) for the semen straws on tiers 1 to 9, while that on tiers 1 to 5 was lower than that on tier 6 to 8 (P < 0.05). GC, freezing rate and the interaction between GC and freezing rate had a significant effect on live sperm numbers (P < 0.01). The highest fertility was in the 6% glycerol group and occurred on day 5 after insemination. The lowest fertility occurred in the 2% glycerol group on day 10 after insemination.

• 4. The optimal combination was 11% glycerol in straws located 6 cm above the LN surface (on tier 6). The 6% glycerol group achieved the highest fertility (77.6%), which surpassed that reported in recent years.

     

Effects of glycerol concentration, equilibration time and temperature of glycerol addition on post-thaw viability of boar spermatozoa frozen in straws

Experiments were conducted to study the effect of glycerol concentration, equilibration time and temperature of glycerol addition on post-thaw viability of boar spermatozoa after cryopreservation in straws. Semen (split ejaculate) in maxi-straws (6 mm o.d.) was frozen using a programmable freezing chamber. Three methods for in vitro sperm evaluation were used: motility (MOT), acrosome integrity (NAR) and flow cytometric analysis of sperm treated with carboxyfluorescein diacetate and propidium iodide to assess sperm plasma membrane integrity (PMI). No interactions were found among the three variables evaluated. Length of prefreeze exposure to glycerol, ranging from .5 min to 75 min, had no effect on post-thaw sperm viability. Exposure of sperm to a glycerol-containing extender medium at 5 degrees C gave improved post-thaw viability over that exposed at 0 degree C (P less than .05). Glycerol at a concentration of 3 or 4% resulted in maximum post-thaw MOT. Acrosome integrity values were greatest for 2 and 3% glycerol, whereas PMI was greatest when glycerol concentration was 4 to 6%. The primary cryoprotective effect of glycerol on boar semen may be extracellular. It is concluded that 3 or 4% glycerol gives maximum viability of frozen-thawed spermatozoa when the present methods are employed.     

Antioxidative effect of BHA in soya bean lecithin‐based extender containing Glycerol or DMSO on freezing capacity of goat semen

The aim of this study was to evaluate the effects of butylated hydroxyanisole (0 or 4 mM) along with different concentrations (5 or 7%) of glycerol (G) and dimethyl sulphoxide (DMSO) as cryoprotectant (CPAs) on freezability of goat semen. Semen was collected from four bucks (3–4 years) twice a week for five weeks. The pooled ejaculates were diluted with extender containing two different concentrations of G or DMSO in combination with BHA. Afterwards, the diluted samples were loaded into 0.25 ml straws and frozen using a standard protocol. After thawing motility parameters, viability, membrane integrity and total abnormality were assessed. The Results showed that the presence of BHA in extender, type and level of CPAs as main factors had significant effects on goat sperm viability, total and progressive motility after freezing–thawing processes (p < .05). Also, the interaction of BHA (0 and 4 mM) and levels of G or DMSO (5 or 7%) had a significant effects (p < .05) on total motility, viability and some characteristic. In this case, the addition of 5% G or DMSO with BHA resulted in highest motility and viability than the other groups (p < .05). The addition of G5 (with and without BHA) increased VSL and reduced abnormality than the other groups (p < .05). The results showed that the main effects of CPAs and CPAs level on membrane functionality were significant (p < .05). Also there were no significance differences in the interactive effects of MDA, VCL, VAP, ALH, LIN and STR among the groups (p > .05). Finally, it can be concluded that the use of 5% CPAs with or without BHA may result in better post‐thaw sperm quality of goat.     

Effects of final dilution rate, sperm concentration and times for cooling and glycerol equilibration on post-thaw characteristics of canine spermatozoa

This study re-evaluated a protocol for cryopreservation of canine semen. Semen from 4 beagle dogs was pooled, concentrated by centrifugation and adjusted to increasing sperm concentrations by adding back seminal plasma. The prepared or original semen was diluted with an extender (Egg yolk-Tris-citrate-glucose) and cooled to 4 degrees C (cooling), followed by a second dilution with the same extender including glycerol, equilibrated at 4 degrees C (equilibration), then stored in liquid nitrogen. The semen was diluted for frozen samples having a fixed sperm concentration with increasing dilution rates or for those having the reverse combinations. Various dilution rates of 2.5-10 folds or sperm concentrations of 0.25-2.5 x 10(8)/ml had no significant effect on post-thaw sperm characteristics. When cooling was done for different times (0-26 hr) with glycerol equilibration for 1 hr, post-thaw characteristics were better at 2 and 3 hr of cooling, while various times for equilibration (0-4 hr) with cooling for 3 hr had no effect. These results suggest that different dilution rates and sperm concentrations within the ranges tested may not affect the post-thaw sperm characterisitics and that sufficient time for cooling may be essential but a specific equilibration time may not necessarily be required.     

Heterosis, direct and maternal genetic effects on semen quality traits of rabbits

A complete diallel cross involving two rabbit sire lines (C and R) was carried out to estimate the crossbreeding genetic parameters of seminal traits. 2140 ejaculates from 153 males were analyzed. The traits studied were: presence of gel plugs (G), urine (U), and calcium carbonate deposits (CC), number of useful ejaculates (UE), pH, volume (V), mass and individual motility (Mm, Mi), useful Mi (UM), concentration (Cn), number of spermatozoa per ejaculate (TSE), percentage of viable spermatozoa (Vi), spermatozoa with normal apical ridge (NAR), normal spermatozoa (Nr), spermatozoa with morphological abnormalities of head (H), neck-midpiece (Nm), and tail (T), presence of proximal and distal cytoplasmic droplet (Dp, Dd).

Estimates of heterosis, direct and maternal genetic effects were obtained from the solutions of the mixed model. There were major differences in direct genetic effects between lines, which were favourable to line C for Cn and TSE, and unfavourable for CC, Nm and Dp. Smaller differences were also observed in Vi and NAR favourable to line R. Differences between lines with respect to the maternal genetic effects were relevant and favourable to line C for V and to line R for U, UM, Cn, TSE, Nm, Mi, and Mm. Individual heterosis was high for Dp and Dd.     

Use of a sperm quality analyser on semen of turkey breeders to monitor storage time effects and age-related changes during a reproductive cycle

1. A relatively new instrument known as a Sperm Quality Analyzer (SQA) offers a rapid assessment of sperm quality and quantity by providing a sperm quality index (SQI). The SQA measures the intensity of sperm activity and motile concentration by determining the number and amplitude of sperm movements per second in a capillary tube as detected through light beam interference. 2. The objectives of the current study were to determine if the SQA could accurately reflect changes in semen quality that occur with prolonged storage of semen and to determine the variation and change in SQI values among individual breeding male turkeys during their semen production cycle. 3. The effect of storage time on SQI values was evaluated by diluting semen with extender and placing the semen on an oscillating shaker at 4 degrees C for 8 h. The SQI values and sperm viability, expressed as % dead sperm, were recorded hourly. The SQI readings declined linearly with increased storage time while % dead sperm increased linearly with increased semen storage. 4. Semen from 220 individual males was analysed monthly for 9 months. Semen diluted 50-fold with saline had lower SQI values during pre- and post-peak phases of production (months 1, 7, 8, and 9 as compared with months 2 to 6 of semen production). The highest SQI values occurred during months 2 to 6. The largest variation in SQI values occurred during months 1 (CV = 26%) and 9 (CV = 31%) with a CV that averaged 16% for the remaining months. 5. Correlation analysis of SQI values for each bird averaged over 9 months with individual male SQIs for each month showed monthly correlation coefficients that ranged from 0.22 to 0.63. 6. These results indicate that the SQA accurately assessed the decline in sperm quality that occurs with prolonged storage of turkey semen and reflected age-related changes in semen quality and quantity that occurred during a semen production cycle of turkey breeders. In addition, the semen quality rank of some turkey breeders in a population changed with age.     

Cryopreservation of boar semen. II: Effect of cooling rate and duration of freezing point plateau on boar semen frozen in mini- and maxi-straws and plastic bags.

The post-thaw motility and the acrosome integrity of semen from 4 boars frozen with a programmable freezing machine, in mini (0.25 ml) and maxi (5 ml) plastic straws and in 10 x 5 cm Teflon FEP-plastic bags (0.12 mm thick, 5 ml), were compared. The freezing of the semen was monitored by way of thermo-couples placed in the straws and the bags. Three freezing programmes were used, namely A: from +5 degrees C, at a rate of 3 degrees C/min, to -6 degrees C, held for 1 min at -6 degrees C, and followed by a cooling rate of 20 degrees C/min to -100 degrees C; B: a similar curve except that there was no holding time at -6 degrees C and that the cooling rate was 30 degrees C/min, and C: from +5 degrees C to -100 degrees C, with a cooling rate of 35 degrees C/min, followed by storage in liquid N2. Despite the freezing curve assayed, both the mini-straws and the bags depicted much shorter freezing point plateaus as compared to the maxi-straws. Post-thaw sperm motility as well as the amount of normal apical ridges were equally significantly higher when semen was frozen in mini-straws or in bags than in maxi-straws. Significant differences in these post-thawing parameters were obtained between the freezing curves used. The stepwise freezing procedure A appeared as the best alternative for boar semen, considering this in vitro evaluation.     

Intrauterine position effects in male and female swine: subsequent survivability, growth rate, morphology and semen characteristics

Pigs of known intrauterine position were obtained from 31 litters by a procedure in which donor sows were slaughtered at d 112 of gestation, their uteri removed and piglets delivered manually. Uterine position was recorded for each piglet as being positioned between two female fetuses (OM), between a male and female fetus (1M), between two male fetuses (2M), between a female fetus and the tip of the uterine horn (OE) or between a male fetus and the tip of the horn (1E). Piglets were fostered as litter groups to recipient sows and reared in these groups until 120 d of age. They then were regrouped and housed as groups of three and six for males and females, respectively. Intrauterine position had no effect on birth weight or survivability of pigs of either sex, although pigs positioned in utero nearest the ovaries (OE and 1E) tended to be heavier at birth. Body weights were similar among groups in each sex at 120 and 175 d of age when given ad libitum access to feed; however, 2M males gained more weight from d 175 to 270 under restricted feeding conditions (P less than .05). Intrauterine position had no effect on anogenital distances either at birth or 120 d of age, and predicted testes weights were similar among males from different positions. Semen characteristics at 220 d of age did not appear to vary due to prenatal environment. Although volume tended to be less for 0M males (P less than .12), concentration, motility and sperm/ejaculate were similar among groups.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of dietary crude protein concentration on semen yield and quality in male broiler breeder fowls

1. The responses in semen volume and spermatozoal concentration of 48 caged male broiler breeder fowls fed on a restricted quantity of food containing 80, 110, 152, 210, 290 or 400 g crude protein (CP)/kg from 18 to 64 weeks of age were studied.

2. The proportion of males producing semen declined with increasing dietary crude protein content and resulted in a significant decrease in total semen volume and spermatozoa production.

3. The relationship between the concentration of spermatozoa in the semen and dietary crude protein was negative but trivial. There were no relationships between dietary crude protein content and semen volume or the metabolic activity of spermatozoa.

4. The metabolic activity of spermatozoa was lower at 25 compared with 37 weeks of age.

5. The proportion of males giving semen peaked between 32 and 42 weeks of age. The relationship between age and semen volume and concentration of spermatozoa was negative and linear.

6. Blood plasma uric acid concentration increased linearly above 106 g CP/kg and males on 400 g CP/kg developed articular gout and had to be destroyed.     

Effect on fertility of low numbers of fowl spermatozoa inseminated in aqueous diluent or semen components of the fowl and turkey

A study was undertaken to compare the effect on fertility in the fowl of aqueous medium, natural homologous seminal plasma, heterologous turkey seminal plasma and whole turkey semen when whole fowl semen was excessively diluted with these media and inseminated fresh. High dilution with fowl seminal plasma resulted in the best fertility. Dilution with the turkey semen components produced fertility no different from that with aqueous diluent when the dose of spermatozoa was 5 X 5 or 10 X 10(6). The results of this study confirm that 5 X 5 to 10 X 10(6) good quality spermatozoa are sufficient to produce acceptable fertility in weekly inseminations of fresh semen. This enables good quality semen to be highly diluted. However, at high dilution rates there is a need to reconsider the composition of semen diluents, with respect to simulating as yet unknown properties provided by factor(s) in homologous ductus deferens seminal plasma.     

Feeding time can alleviate negative effects of heat stress on performance,meat quality and health status of turkey

1. A total of 180 one-day-old turkeys were randomly assigned to 6 equal groups to investigate the effect of feeding time on growth performance, carcass characteristics, meat quality, leg problems and physiological responses of growing turkeys under the high temperature conditions of summer.

2. Birds of the first group were ad libitum fed and were considered as the controls (C). The second group (T1) was given 80% of diet in the morning and 20% of diet in the afternoon, the third group (T2) was given 60% of diet in the morning and 40% of diet in the afternoon, the fourth group (T3) was given 40% of diet in the morning and 60% of diet in the afternoon, the fifth group (T4) was given 20% of diet in the morning and 80% of diet in the afternoon and the sixth group (T5) was given 100% of diet in the afternoon.

3. Body weight, body weight gain and feed conversion ratio were improved with T2, T3, T4 and T5 in comparison to control or T1 under heat stress conditions. No significant impacts on carcass traits and meat quality due to changing the time of feeding were seen, except for tenderness and juiciness.

4. Feeding in the afternoon (100%) decreased body temperature and tonic immobility test score, which were positively related with the health condition of the birds.

5. The incidence of leg problems, plumage condition and breast blisters were not significantly different among the experimental groups.

6. It is concluded that feeding turkeys mainly or totally in the afternoon (T4 and T5, birds were fed with 80% or 100% of the diet in the afternoon) can be used as a strategy and a managerial tool for improving growth rate, feed utilisation, carcass and meat quality, as well as health status of growing turkeys reared under hot climate conditions.