K. Buranaamnuay

Effects of Straw Volume and Equex-STM® on Boar Sperm Quality after Cryopreservation

The present experiments were designed to study the effect of adding the detergent Equex-STM to freezing extender, and of straw volume (0.25 ml vs 0.5 ml), on boar sperm quality after cryopreservation. Three ejaculates from each of four purebred boars (three Landrace and one Yorkshire) were collected and frozen with a lactose-egg yolk extender containing glycerol with or without 1.5% Equex-STM. The extended semen was loaded into either 0.25- or 0.5-ml straws. The straws were placed in liquid nitrogen (LN(2)) vapour approximately 3 cm above the level of LN(2) for 20 min and then were plunged into LN(2). Thawing was achieved in warm water at 50 degrees C for 12 s and then was incubated in a 38 degrees C water-bath for 30 min before evaluating sperm quality. Results showed that the individual motility, viability and acrosomal normal apical ridge (NAR) were improved (p < 0.001) when Equex-STM was added to the freezing extender. There was no difference (p = 0.48) in sperm motility between 0.25- and 0.5-ml straws when Equex-STM was added. The percentages of viable and of NAR sperm in 0.5-ml straws were higher than those in 0.25-ml straws (p = 0.02, p = 0.0003 respectively). The percentages of membrane intact sperm evaluated using the short hypo-osmotic swelling test were not affected by straw volume or the adding of Equex-STM (p > 0.05). The results of these investigations suggested that Equex-STM exerts a beneficial effect on the quality of cryopreserved boar semen and this cryopreservation protocol was favourable for a 0.5-ml straw.

Assessment of motility of ejaculated, liquid-stored boar spermatozoa using computerized instruments

Visual-motility assessment is a tool used to determine the quality of boar ejaculates. This method is subjective by nature, and consequently, computer-assisted sperm analysis (CASA), with different software designs, has been developed for more objective assessment using conventional image analysis or particle counting. In the present study, we compared the results of sperm analysis using a conventional CASA system (Cell Motion Analyzer, SM-CMA), with those using a novel software (QualiSperm) and those of visual assessment performed by two operators. Ejaculates were collected weekly from four Swedish Landrace boars for 4 weeks. Each ejaculate was divided into three aliquots of different sperm concentration (300, 125, and 40 million spermatozoa/mL) and stored at approximately 17 degrees C for 96h. Only samples at 40 million spermatozoa/mL concentration were analyzed using both automated systems; for the remaining concentrations, the SM-CMA was not used due to its inability to examine higher sperm concentrations. The number of spermatozoa analyzed was highest for the QualiSperm ( approximately 300-5000 spermatozoa), followed by the SM-CMA ( approximately 200 spermatozoa), and lastly, by subjective motility evaluation ( approximately 100 spermatozoa). There was a time-course decrease in motility of the liquid-stored semen, detectable by either computerized method. Although the percentage of motile spermatozoa measured by the two automated systems correlated well (r > or = 0.75), there was disagreement between operators. In conclusion, because of the lower degree of variation, the numbers of spermatozoa analyzed, and the speed of analysis ( approximately 1 min per sample), QualiSperm appears to be a suitable instrument for routine work, provided it maintains stability and is available at an affordable price.

Influence of cryoprotectants glycerol and amides, combined with antioxidants on quality of frozen-thawed boar sperm

The present study was undertaken to examine whether the cooling and freezing extenders containing a mixture of antioxidants (AOs) catalase, Na-pyruvate and mercaptoethanol and one of three types of cryoprotectants (CPs) would be able to improve the quality of frozen-thawed boar sperm. The collected semen, only the sperm-rich fraction, was diluted 1:1 with Androhep plus™ extender, stored at 15°C for 2 h and centrifuged. The centrifuged sperm pellet was re-suspended in lactose-egg yolk extender and divided into four groups for mixing with freezing extenders containing different kinds of CPs at 5°C: (I) glycerol (GLY) as control; (II) GLY with AOs; (III) dimethyl formamide (DMF) with AOs and (IV) dimethyl acetamide (DMA) with AOs. Processed sperm were packaged in 0.25-mL straws and frozen using a controlled rate freezer. After thawing, the diluted thawed sperm were incubated at 38°C for 10 min and was assessed for motility by CASA, membrane/acrosome integrity by FITC-PNA/PI and DNA integrity (DFI) by SCSA. All sperm parameters evaluated, except DFI, were negatively affected (P<0.001) when using DMF (III) or DMA (IV) as CPs instead of GLY (I and II). Total sperm motility was lower (P<0.001) in the samples supplemented with AOs (32.4 ± 1.2, 23.9 ± 1.5, 6.9 ± 0.7, and 10.3 ± 0.9%, for treatments I, II, III and IV, respectively). The quality of sperm frozen in DMF was not different from DMA (P>0.05). There was no difference in DFI among the studied groups (P>0.05). In conclusion, based on the present results, addition of AOs to cooling and freezing extenders and/or replacement of GLY with DMF or DMA could not improve quality of frozen-thawed boar sperm.

Protocols for sperm cryopreservation in the domestic cat: a review

The main objectives of sperm cryopreservation in domestic cats are to preserve these gametes for future use, especially in valuable domestic cat breeds and to use knowledge-gained for developing sperm preservation techniques in wild felids that are threatened with extinction. To achieve acceptable quality of post-thaw sperm and results after insemination, sperm samples must be properly handled, cryopreserved and thawed by using appropriate protocols. In this paper, cryopreservation protocols of domestic cat sperm that have been reported previously are described. The subtopics include sources of sperm, freezing extenders, methods of sperm dilution, freezing storage vessels, methods of sperm cryopreservation, thawing temperature, and thawing extenders. In addition, comparisons of sperm quality results for different treatments within the same studies and between different studies are also presented.

Preliminary study on effects of bovine frozen semen storage using a liquid nitrogen-independent method on the quality of post-thaw spermatozoa

Frozen semen of eight bulls was used to assess effects of storage temperature and length of storage time on frozen-thawed bovine sperm quality. In experiment 1, 25 straws of frozen semen of each bull were allocated to 3 groups. The control was still maintained in liquid nitrogen (LN2). The rest were abruptly moved from LN2 to -80°C and -30°C mechanical freezers, respectively. After thawing, it was found that the sperm motility, vitality and membrane integrity were comparable (P>0.05) between the control and the -80°C samples and were significantly inferior (P<0.001) in the -30°C samples, irrespective of storage time (1-day, 1-week and 1-month). In experiment 2, two out of the three parts (16-18 straws) of frozen semen of each bull were rapidly removed from LN2 and further kept in the freezer (-80°C). One day before being thawed, half of the samples in the freezer were promptly put back to LN2. The results showed that the frozen-thawed sperm quality was not significantly affected (P>0.05) both by storage temperature (-196°C, -80°C and -80 & -196°C) and storage time [day-2, day-8 (1-week) and day-31 (1-month)]. At the same storage times, the quality measures at different temperatures were not significantly different from one another (P>0.05). In conclusion, a -80°C mechanical freezer was as effective as LN2 in preserving in vitro quality of frozen-thawed bovine spermatozoa throughout 1-month of storage. When required for use, frozen semen stored in the freezer could be thawed immediately or transferred to the LN2 tank for thawing elsewhere.

The in vitro quality of frozen-thawed Asian elephant (Elephas maximus) spermatozoa in semen supplemented with Equex STM paste and oxytocin during and after cryopreservation

The effects of Equex STM paste (Equex) and oxytocin (OT) on the in vitro quality of frozen-thawed Asian elephant sperm were investigated in the study. The viability of frozen-thawed sperm was significantly higher in the Equex-treated (1 and 2%) groups than in the control group. There were no differences in the examined sperm parameters among the control and OT-treated (0.05-5IU) groups.

Improvement of Semen Quality by Feed Supplement and Semen Cryopreservation in Swine

Artificial insemination in pig offers many advantages in swine production in terms of a better disease control through semen quality control, a diverse male genetic distribution and an easiness of management. It is accepted that in developing countries, AI helps to improve the genetic profile. A number of sows can be inseminated using the same ejac‐ ulate instead of only one from natural mating. The number of pig farms using AI has increased because of the technical improvement of semen extenders and equipments, and the technique can be performed on farm. In Thailand, AI in commercial pig farms is routinely used as a standard protocol in pig production. The results obtained by AI are quite similar or higher than that from natural. Because of the quality of insemina‐ tion can be guaranteed by semen testing and evaluation before insemination. The im‐ provement of semen quality can be acquired by feed supplement and semen freezing in boar can be used to genetic conservation. The feed supplement improving the semen quality have been imperatively used in the boars which have low libido and low se‐ men quality, because these boars have been imported and are of superior genetic merit and so are perceived to have great value to their owners who, therefore, are very reluc‐ tant to cull them. Moreover, in tropical countries, cryopreservation of boar semen is nowadays performed in a limited scale and it has yet to be conducted in Thailand par‐ ticularly for the commercial purpose. Concerning this point and obtained benefit in the future, the improvement of boar semen quality by feed supplement and boar semen cryopreservation are reviewed in this chapter.

Fertilization Rate and Number of Embryos on Day 2 after Intrauterine and Deep Intrauterine Insemination Using Frozen-Thawed Boar Semen in Multiparous Sows

The present study determines fertilization rate and number of embryos on Day 2 after intrauterine insemination (IUI) and deep intrauterine insemination (DIUI) using frozen-thawed (FT) boar semen in multiparous sows. Twelve crossbred Landrace × Yorkshire multiparous sows were included. The sows were inseminated at 24 h after oestrus detection and reinseminated every 12 h until ovulation took place. The inseminations were conducted using IUI with 2 × 109 FT sperm per dose (n = 6) and DIUI with 1 × 109 FT sperm per dose (n = 6). The sows were slaughtered at 45.1 ± 7.2 h after ovulation. Embryos and unfertilized oocytes were flushed from the oviducts. IUI yielded a better fertilization rate than DIUI (66.0% versus 31.0%, P < .001). The number of embryos was 13.5 ± 2.7 and 6.6 ± 3.2 embryos/sow in IUI and DIUI groups, respectively (P = .08). The proportion of sows having unilateral fertilization was higher in the DIUI (3/5) than the IUI group (1/6). In conclusion, IUI with at least 2 × 109 total number of FT boar spermatozoa is recommended.

Cryopreservation and storage of cat epididymal sperm using ‒75 °C freezer vs liquid nitrogen

The quality of cat epididymal sperm cryopreserved and stored by four methods was assessed. Epididymal sperm were suspended in Tris-glucose-citrate egg yolk extender, loaded in 0.25 mL straws and then cryopreserved. The samples in a standard protocol (LN) were cryopreserved and stored in liquid nitrogen (LN2). The sperm straws in the LN-Fr-LN group were cryopreserved in LN2 and stored in a -75 °C freezer; the straws were returned to LN2 prior to thawing. The loaded straws in the Fr group were transferred directly from 4 °C to the freezer and maintained in the freezer until thawing. The Fr-LN samples were cryopreserved and stored in the freezer and were introduced into LN2 before thawing. The sperm thawing was conducted on days 30, 60, 90 and 120 of cryopreservation. The sperm motility, viability, membrane integrity and acrosome integrity were evaluated at 15 and 180 min after thawing. The quality of post-thaw sperm in all three modified protocols was comparable (P > 0.05) and did not differ from that in the standard protocol except the membrane integrity of the 60 days stored samples evaluated at 15 min after thawing, which was significantly higher for the LN-Fr-LN than the Fr-LN groups (P = 0.04). The length of cryopreservation time had no effect (P > 0.05) on the sperm parameters assessed at 15 min after thawing. The sperm motility was significantly greater (P = 0.01 to P = 0.02) for the 15 min than the 180 min incubation. In conclusion, cat epididymal sperm could alternatively be cryopreserved and/or stored by using the -75 °C freezer for 120 days. To use, the cryopreserved sperm in the freezer could be thawed immediately or after being transferred to LN2. This was useful for the application of the -75 °C cryopreserved sperm in remote areas.