Mongkol Techakumphu

The effect of antioxidants on motility, viability, acrosome integrity and DNA integrity of frozen-thawed epididymal cat spermatozoa

Antioxidants partially ameliorated the negative effects of reactive oxygen species (ROS) produced during cryopreservation. The objective of the present study was to investigate the effect of cysteine and a water-soluble vitamin E analogue on the quality of frozen-thawed epididymal cat spermatozoa. Epididymal spermatozoa were collected from eight male cats and divided into three aliquots; these were resuspended with a tris egg yolk extender I (EE-I), or the same extender supplemented with 5mM dl-cysteine (EE-C) or with 5mM of a water-soluble vitamin E analogue (EE-Ve). Prior to the freezing step, sperm suspensions were added to the extender with Equex STM paste (EE-II). Sperm motility, progressive motility, membrane integrity, and acrosome status were evaluated at collection, after cooling, and at 0, 2, 4, and 6h post-thaw. Sperm DNA integrity was evaluated at 0 and 6h post-thaw. Relative to the control group, supplementation with vitamin E improved (P<0.05) post-thaw motility (69.4+/-5.6%), progressive motility (3.9+/-0.3), and membrane integrity (65.1+/-8.1%) immediately after thawing, whereas cysteine supplementation improved (P<0.05) post-thaw motility after 2h of incubation (53.8+/-12.2%) and DNA integrity after 6h (84.1+/-4.4%). However, neither antioxidant significantly increased the acrosome integrity of frozen-thawed spermatozoa. In conclusion, cysteine or vitamin E supplementation of tris egg yolk extender improved motility, progressive motility and integrity of the sperm membrane and DNA of frozen-thawed epididymal cat spermatozoa.

Lipid profiles of sperm and seminal plasma from boars having normal or low sperm motility

Sperm plasma membrane lipids have an important role to play in determining membrane fluidity and sperm motility. The objective of the present study was to determine whether there are differences in the lipid and fatty acid (FA) composition of boar sperm and seminal plasma in the ejaculates of boars having different sperm motilities. Semen was collected from two groups of boars having normal (> 60%; n = 53) or low (< 60%; n = 53) motility sperm and the semen was evaluated for motility, morphology and vitality. The semen was then centrifuged to separate the sperm from the seminal plasma and both were kept at -20 °C until analyzed for lipid content and FA profile by gas chromatography. Total antioxidant status (TAS) of seminal plasma was determined using a commercial kit. There were differences (P ≤ 0.05) in sperm total lipids, cholesterol, saturated fatty acids (SFA), phospholipids, n-3 polyunsaturated fatty acids (PUFA), docosahexaenoic acid (DHA) and the ratio of n-6:n-3 PUFA between boars with normal and low motility sperm. Total lipids, cholesterol, phospholipids, PUFA, DHA and n-3 PUFA were positively correlated with sperm motility, viability, normal morphology and normal plasma membrane. In contrast, SFA and the ratio of n-6: n-3 PUFA were negatively correlated (P ≤ 0.05) with sperm motility, viability, normal morphology and normal plasma membranes. The TAS of seminal plasma from boars having normal motility sperm was higher (P ≤ 0.05) than that of boars having low motility sperm and TAS was positively correlated (P = 0.0001) with sperm motility, viability, normal morphology and normal plasma membranes. In summary, differences in sperm motility were related to n-3 PUFA content in the sperm plasma membrane and extracellular antioxidants in seminal plasma which protect sperm plasma membranes from lipid peroxidation during periods of oxidative stress.

Embryoid body formation from embryonic and induced pluripotent stem cells: Benefits of bioreactors

Embryonic stem (ES) cells have the ability to differentiate into all germ layers, holding great promise not only for a model of early embryonic development but also for a robust cell source for cell-replacement therapies and for drug screening. Embryoid body (EB) formation from ES cells is a common method for producing different cell lineages for further applications. However, conventional techniques such as hanging drop or static suspension culture are either inherently incapable of large scale production or exhibit limited control over cell aggregation during EB formation and subsequent EB aggregation. For standardized mass EB production, a well defined scale-up platform is necessary. Recently, novel scenario methods of EB formation in hydrodynamic conditions created by bioreactor culture systems using stirred suspension systems (spinner flasks), rotating cell culture system and rotary orbital culture have allowed large-scale EB formation. Their use allows for continuous monitoring and control of the physical and chemical environment which is difficult to achieve by traditional methods. This review summarizes the current state of production of EBs derived from pluripotent cells in various culture systems. Furthermore, an overview of high quality EB formation strategies coupled with systems for in vitro differentiation into various cell types to be applied in cell replacement therapy is provided in this review. Recently, new insights in induced pluripotent stem (iPS) cell technology showed that differentiation and lineage commitment are not irreversible processes and this has opened new avenues in stem cell research. These cells are equivalent to ES cells in terms of both self-renewal and differentiation capacity. Hence, culture systems for expansion and differentiation of iPS cells can also apply methodologies developed with ES cells, although direct evidence of their use for iPS cells is still limited.

Supplementation of maturation medium with L-carnitine improves cryo-tolerance of bovine in vitro matured oocytes

The objective was to determine the effects of adding L-carnitine (an enhancer of lipid metabolism) during IVM, on cryotolerance and developmental competence of bovine oocytes. Oocytes matured in the absence (control) or presence (0.6 mg/mL) of L-carnitine were subjected to IVF and embryo culture after Cryotop vitrification or nonvitrification at the metaphase stage of the second meiotic cell division. Cleavage and blastocyst formation rates, and inner cell mass and trophectoderm cell numbers were determined. Also, ATP content in IVM oocytes was measured and intracellular lipid droplets were observed (Nile red staining and confocal microscopy). L-carnitine had no significant effect on the rate of matured oocytes. Vitrification reduced (P < 0.05) mean (±SEM) rates of live oocytes both in control (80.6 ± 1.9%) and L-carnitine groups (82.7 ± 5.1%) compared with nonvitrified oocytes (100%). After IVF, cleavage rates of vitrified control and L-carnitine groups (56.5 ± 3.9% and 62.8 ± 5.1%, respectively) were significantly lower than those in nonvitrified control and L-carnitine groups (83.9 ± 4.2% and 84.3 ± 1.3%). After vitrification, blastocyst formation rate in the L-carnitine group (54.4 ± 5.2%) was significantly higher compared with the control (34.9 ± 4.4%), and did not significantly differ from those in nonvitrified control and L-carnitine groups (52.1 ± 4.2% and 52.8 ± 3.0%). The numbers and ratio of inner cell mass and trophectoderm cells in blastocysts did not differ significantly among groups. The ATP content in L-carnitine-treated oocytes tended to be higher compared with the control. Vitrification did not reduce ATP content in oocytes, irrespective of L-carnitine treatment. Treatment with L-carnitine dislocated lipid droplets from the peripheral area to the inner cytoplasm. In conclusion, L-carnitine supplementation during IVM redistributed lipid droplets in oocytes; if they survived vitrification, their developmental competence was similar to that of nonvitrified oocytes.

Effect of docosahexaenoic acid on quality of cryopreserved boar semen in different breeds.

During the cryopreservation process, the level of polyunsaturated fatty acids, especially docosahexaenoic acid (DHA), in the sperm plasma membrane decreases significantly because of lipid peroxidation, which may contribute to sperm loss quality (i.e. fertility) of frozen-thawed semen. The aim of this study was to investigate the effect of supplementation of DHA (fish oil) in freezing extender II on frozen-thawed semen quality. Semen from 20 boars of proven motility and morphology, were used in this study. Boar semen was split into four groups, in which the lactose-egg yolk (LEY) extender used to resuspend the centrifuged sperm pellet was supplemented with various levels of fish oil to reach DHA level of 1X (group I, control, no added fish oil), 6X (group II), 12X (group III) and 18X (group IV). Semen solutions were frozen by using a controlled rate freezer. After cryopreservation, frozen semen was thawed and evaluated for progressive motility, viability by using SYBR-14/Ethidiumhomodimer-1 (EthD-1) staining and acrosome integrity by using FITC-PNA/EthD-1 staining. There was a significantly higher (p < 0.001) percentage of progressive motility, viability and acrosome integrity in DHA (fish oil) supplemented groups than control group. Generally, there seemed to be a dose-dependent effect of DHA, with the highest percentage of progressive motility, viability and acrosome integrity in group-III. In conclusion, supplementation of the LEY extender with DHA by adding fish oil was effective for freezing boar semen as it resulted in higher post-thaw plasma membrane integrity and progressive motility.

Supplemental effect of varying L-cysteine concentrations on the quality of cryopreserved boar semen.

Cryopreservation is associated with the production of reactive oxygen species, which leads to lipid peroxidation of the sperm membrane and consequently a reduction in sperm motility and decreased fertility potential. The aim of this study was to determine the optimal concentration of L-cysteine needed for cryopreservation of boar semen. Twelve boars provided semen of proven motility and morphology for this study. The semen was divided into four portions in which the lactose-egg yolk (LEY) extender used to resuspend the centrifuged sperm pellet was supplemented with various concentrations of L-cysteine to reach 0 mmol L(-1) (group I, control), 5 mmol L(-1) (group II), 10 mmol L(-1) (group III) and 15 mmol L(-1) (group IV). Semen suspensions were loaded in straws (0.5 mL) and placed in a controlled-rate freezer. After cryopreservation, frozen semen samples were thawed and investigated for progressive motility, viability using SYBR-14/EthD-1 staining and acrosome integrity using FITC-PNA/EthD-1 staining. There was a significantly higher (P < 0.01) percentage of progressive motility, viability and acrosomal integrity in two L-cysteine-supplemented groups (group II and group III) compared with the control. There was a biphasic effect of L-cysteine, with the highest percentage of progressive motility, viability and acrosomal integrity in group III. In conclusion, 5 or 10 mmol L(-1) was the optimum concentration of L-cysteine to be added to the LEY extender for improving the quality of frozen-thawed boar semen.

Seasonal influences on the litter size at birth of pigs are more pronounced in the gilt than sow litters

The aim of the present study was to use data from herds to demonstrate the degree of seasonal influence on litter size at birth in gilts compared to sow parities 2, 3–5 and older (parities ⩾6) in a conventional, open-housing system for commercial pig herds in the northeastern part of Thailand. Data were obtained during a 3-year period from July 2005 to June 2008. The data analysed included observations on 25 835 litters from 8100 sows. Total number of piglets born per litter (TB), number of piglets born alive per litter (BA), proportion of stillborn piglets per litter (SB) and proportion of mummified fetuses per litter (MF) were analysed using a general linear mixed model procedure. The influence of temperature, relative humidity and temperature-humidity index (THI) on TB, BA, MF and SB were also analysed. The meteorological data were merged with the reproductive data and the means of temperature, relative humidity and THI during 115 days before farrowing were calculated and included in the statistical models. The results revealed that sows that farrowed in the hot season had a larger TB and BA than sows that farrowed in the rainy ( P P P P =0·01), 0·3 ( P =0·003) and 0·3 ( P =0·02) TB fewer than those that farrowed in the hot season. In the first parity, MF increased from 0·022 to 0·042 when the mean temperature during gestation increased from 26 to 29°C ( P P P P P >0·05). In conclusion, inferior litter size at birth was observed in sows that farrowed in either rainy or cool seasons. High temperature, high relative humidity and/or high THI during gestation significantly reduced the number of total piglets born per litter. The influence of season, temperature, relative humidity and/or THI on litter size at birth was more evident in the gilts than the sows. These data indicated that various strategies to reduce temperature in the open-housing system for pregnant gilts and sows in Thailand are not adequate and the proper housing of pregnant gilts should be emphasized.

The association between growth rate, body weight, backfat thickness and age at first observed oestrus in crossbred Landrace × Yorkshire gilts

The present study aims to investigate the association between growth rate (GR), body weight (BW), backfat thickness (BF) and age at first observed oestrus in crossbred Landrace x Yorkshire (LY) replacement gilts in the tropics. The study was carried out on five commercial swine herds in Thailand between 2004 and 2006. A total of 6946 LY gilts were included. The gilts entered the herd at about 163 days of age. The BW (kg) and BF (mm) of the gilts were measured when the gilts entered the gilt pools and again when the gilts were sent to the breeding house. The GR from birth to entry into the gilt pools (birth to 90 kg BW) (GRe), the GR from entry into to exit from the gilt pools (91-134 kg BW) (GRi) and the GR from birth until the gilts were sent to the breeding house (birth to 134 kg BW) (GRs) were calculated. The relationship between age at first observed oestrus and GRe, GRs, GRi, BW and BF were analyzed. Pearsons correlation and four general linear models (GLMs) were conducted. On average, the gilts showed first observed oestrus at 200+/-28 days of age. The means of age at first observed oestrus varied from 188 to 251 days (P<0.001) among the herds. The GRs of the gilts significantly correlated with the BW (r=0.55, P<0.001) of the gilts when they were sent to the breeding house and the age at first observed oestrus (r=-0.40, P<0.001). Gilts with a high GRe and GRs were younger at first observed oestrus compared to gilts with a low GRe and GRs. On average, the gilts with GRs of over 604 g/day showed first observed oestrus before 5 months of age. GRi was not correlated with the age at first observed oestrus (P>0.05). Neither the BF of the gilts at entry nor the BF that the gilts gained within the gilt pools significantly correlated with age at first observed oestrus (P=0.29 and P=0.69, respectively). But the gilts with a higher BF at entry tended to have a higher BW when they were sent to the breeding house (r=0.44, P<0.001). The present study indicates that replacement gilts with a high GR (both GRe and GRs) tend to show sign of oestrus earlier than gilts with a low GR (both GRe and GRs).

Influence of repeat-service and weaning-to-first-service interval on farrowing proportion of gilts and sows.

The present study was performed to evaluate different components of reproductive failure after service under a tropical climate and to investigate the influence of repeat-service and delayed wean-to-service interval (WSI) on subsequent fertility in gilts and sows. The study was conducted in four commercial swine breeding herds in the northeastern part of Thailand. Data were collected during a 3-year period from July 2005 to June 2008. A total of 30,058 insemination records from 9037 gilts and sows was included. On average, the farrowing proportion (FP) was 81.9% and adjusted FP (excluding gilts/sows culled after service) was 85.3%. The reasons for the failure to farrow included return-to-oestrus 9.4%, abortion 1.7%, not being pregnant 1.0% and not-in-pig 2.0%. Non-repeat-service females had 83.7% FP, while those that experienced repeat-serviced for 1, 2 and > or = 3 times had 71.2%, 57.7% and 43.4% FP, respectively (P<0.001). The seasonal influence on FP was observed in non-repeat-serviced females, but not in those that experienced repeat-service. Sows mated during 0-6 days after weaning had 86.8% FP, while those mated 7-10, 11-20 and 21-60 days after weaning had 78.9%, 78.9% and 78.4% FP, respectively (P<0.001). It is concluded that repeat-service in gilts/sows resulted in a 12.5% decrease in FP. Sows returning to oestrus later than 6 days after weaning had 7% lower FP than sows mated within 6 days after weaning.

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.