Cryopreservation is a widely-used efficient means of long-term sperm preservation. However, unlike other types of semen, cryopreserved boar semen has reduced fertility and the efforts continue to optimize post-thawing sperm recovery. In this study, we evaluated the effects of various washing solutions (Hulsen solution, labmade DPBS and commercial DPBS) on post-thawing porcine sperm kinematics (CASA system), viability (SYBR-14/PI) and acrosome integrity (PSA/FITC). We also examined the effect of washing-centrifugation on frozen-thawed semen kinematics. The results indicate that type of washing solution and post-thawing centrifugation alters parameters linked to sperm quality (total motility, progressive motility, viability and acrosome integrity). Significantly higher (p < 0.05) motility and progressive motility were obtained when cryopreserved semen was processed with Hulsen solution. The postthaw percentage of live and intact acrosomal sperm was significantly higher in group 1 (Hulsen solution) as compared to other groups. Following thawing-centrifugation, the results showed significantly higher motility and progressive motility in group 1 than other groups. However, the latter two DPBS groups did not differ statistically. Taken together, Frozen-thawed spermatozoa motility, acrosome integrity and viability can be affected by the type of washing solution used. Moreover, centrifugation of frozenthawed semen has an unfavorable effect on total motility and progressive motility.
Halla horse is crossbreed between Jeju and Thoroughbred horses and is used for riding, racing and meat production. Thus, molecular genetic studies are needed to establish and preserve the industrially valuable Halla horses. This study aimed to analyses the genetic diversity and population structure through 12 microsatellite (MS) markers for Halla and putatively related 3 breeds (Jeju, Mongolian and Thoroughbred horses). On average, the number of alleles, observed heterozygosity (Hobs), expected heterozygosity (Hexp), and polymorphic information content (PIC) among all horses were 10, 0.767, 0.799, and 0.771, respectively. Neighbor-joining tree and STRUCTURE analysis showed that Halla horses were between Thoroughbred and Jeju horses, tend to more influenced by Thoroughbred horses. Therefore, these results could be considered for use as the basic genetic breed relationships resource among the horse breeds (Jeju, Mongolian, and Thoroughbred horses) related to the origins of the Halla horse.
In the present study, we examined if deep uterine artificial insemination (DUAI) can improve the pregnancy rate of artificial insemination (AI) using epididymal spermatozoa (ES) in Hanwoo cattle. The estrus cycles of 88 Hanwoo cows were synchronized, and 17 cows were artificially inseminated using the DUAI method with ES, 20 cows were artificially inseminated via the uterine body (BUAI) method with ES, and as a control, 51 cows were inseminated by using the BUAI method with ejaculated spermatozoa from 1 proven bull after frozen thawing. The pregnancy rate of the DUAI method (58.8%) was higher than that of the BUAI method (25.0%, p = 0.0498). The motility of ES was examined immediately after thawing and after 3 and 6 h of incubation. The rapid progressive sperm motility of the control group was significantly higher than that of the ES group immediately after thawing and after 3 and 6 h of incubation (p < 0.05). The straight line velocity and average path velocity of the ES group after 6 h of incubation were significantly lower than those in the control group (p < 0.05). The linearity and amplitude of lateral head of ES were lower than those at 6 h (p < 0.05). The flagellar beat cross frequency and hyperactivation of ES were lower than the control spermatozoa immediately after thawing and at 3 h (p < 0.05). These motility parameters suggested that ES had a low motility and fertilization ability compared to the control spermatozoa. After frozen-thawing and 3 h of incubation, the percentage of live spermatozoa with intact acrosomes in the ES was significantly lower than that in ejaculated spermatozoa (p < 0.05). Our findings suggested that the DUAI method can overcome the low pregnancy rate of ES, despite the low motility, viability, and fertilization ability of ES.
The objective of this study was to establish a selection process for high quality sperm in bovine semen using sperm separation by magnetic activation (MACS). For this, semen from 21 Nellore bulls was collected using an artificial vagina. To guarantee the presence of pathologies in the ejaculate, animals previously declassified in four consecutive spermiogram were used. Semen was analyzed in five statuses: (1) fresh semen (fresh); (2) density gradient centrifugation (DGC), percoll column; (3) non-apoptotic fraction after separation by MACS (MAC); (4) apoptotic fraction from the separation (MACPOOR); and (5) MAC followed by DGC (MACDGC). Using a computerized analysis system (CASA), motility was measured. The sperm morphology was evaluated by phase contrast, and the supravital test was completed with eosin/nigrosin staining. For DGC, 20 × 106 cells were used in a gradient of 90% and 45% percoll. MACS used 10 × 106 cells with 20 μL of nanoparticles attached to annexin V, and filtered through the MiniMACS magnetic separation column. Membrane integrity was assessed with SYBR-14/IP and mitochondrial potential with JC-1 by flow cytometry. Processing sperm by MACDGC, was more effective in obtaining samples with high quality sperm, verified by the total of abnormalities in the samples: 35.04 ± 2.29%, 21.50 ± 1.47%, 17.30 ± 1.10%, 30.68 ± 1.94% and 10.50 ± 1.46%, respectively for fresh, DGC, MAC, MACPOOR, and MACDGC. The subpopulation of non-apoptotic sperm had a high number of live cells (82.65%), membrane integrity (56.60%) and mitochondrial potential (83.98%) (p < 0.05). These findings suggest that this nanotechnological method, that uses nanoparticles, is efficient in the production of high-quality semen samples for assisted reproduction procedures in cattle.
In horse management, the alarm system with sensors in the foaling period enables the breeder can appropriately prepare the time of the parturition. It is important to prevent losses by unpredictable parturition because there are several high risks such as dystocia and the death of foals and mares during foaling. However, unlike analysis in the alarm system that detects specific motions has been widely performed, analysis of classification following specific behavior patterns or number needs to be more organized. Thus, the objective of this study is to classify signs of the specific behaviors of the mares for the prediction of pre-foaling behaviors. Five Thoroughbred mares (9-20 yrs) were randomly selected for observation of the prefoaling behaviors. The behaviors were monitored for 90 min that was divided into three different periods as 1) from -90 to -60 min, 2) from -60 to -30 min, 3) from -30 min to the time for the discharge of the amniotic fluid, respectively. The behaviors were divided into two different categories as state and frequent behaviors and each specific behavioral pattern for classification was individually described. In the state behaviors, the number of mares in the standing of the foaling group (3.17 ± 0.18b) at period 3 was significantly higher than the control group (1.67 ± 0.46a). In contrast, the number of the mares in the eating of the foaling group (1.17 ± 0.34b) at period 3 was significantly lower than the control group (3.33 ± 0.46a). In the frequent behaviors, the weaving of the foaling group was significantly higher than the control group, and looking at the belly of the foaling group was significantly lower than the control group. In period 2, defecation, weaving, and lowering the head of the foaling group were significantly higher than the control group, respectively. In period 3, sitting down and standing up, pawing, weaving, and lowering the head in the foaling group were also significantly higher than the control group. In conclusion, the behavior is significantly different in foaling periods, and the prediction of foaling may be feasible by the detection of the pre-foaling behaviors in the mares.
Sperm cryopreservation is an important method of assisted reproductive techniques and storing genetic resources. It plays a vital role in genetic improvement, livestock industrial preservation of endangered species, and clinical practice. Consequently, the cryopreservation technique is well organized through various studies, especially on Korean native cattle (Hanwoo). However, the cryopreservation technique of Korean native brindled cattle, which is one of the native cattle species in Korea, is not well organized. Therefore, it is necessary to develop a Supplementary Table technique for the cryopreservation of Korean native brindled cattle. For this purpose, it is important to first evaluate the quality of the currently produced cryopreserved sperm of Korean native brindled cattle. In this study, we randomly selected 72 individual Korean native brindled cattle semen samples collected from 8 different region research centers and used them to evaluate sperm functions. We focused on the quality evaluation of cryopreserved Korean native brindled cattle semen following the measurement of motion kinematics, capacitation status, intracellular ATP level, sperm motility, and cell viability. Then, the values of each of the eight groups were derived from various sperm parameters of nine individual samples, including sperm motility, kinematics, cellular motility, and intracellular ATP levels, which were used to compare and evaluate sperm function. Overall, differences in various sperm parameters were observed between most of the research centers. Particularly, the deviations of motility and motion kinematics were high according to the sample. Therefore, we suggest that it is necessary to develop a standard method for the cryopreservation of Korean native brindled cattle semen. We also suggest the need for sperm quality evaluation of the cryopreserved semen of Korean native brindled cattle before using artificial insemination to attain a high fertility rate.
Three different cats who had chronic kidney disease (CKD) were treated for more than one month with fluid therapy in an animal clinic. Although this long-term treatment and hospitalization, there was no clinical improvement in clinical signs as well as serum biochemical indexes including blood urea nitrogen (BUN), creatinine (CREA), and phosphate (PHOS). All cases were then injected three times with allogeneic stem cells through an intravenous route for treatment on Day 0, 7, and 14 or 30. On the same day, clinical observation and blood tests for serum biochemistry were conducted together. Upon administrating stem cells to the CKD cats, clinical conditions and the indexes of BUN and CREA were clinically improved within normal ranges. Additionally, one of the cats who had the renal cysts presented clinical improvement with showing decreased cysts size than before.