The effects of the number of frozen-thawed ram sperm per single and double intra-cervical artificial insemination (AI) on fertility in ewes were studied. A total of 89 non-pregnant ewes were synchronized for oestrus with two doses of 100 μg PGF2α (Cloprostenol) 9 days apart. The ewes were randomly assigned to one of four groups; D200 (n = 23; double AI with 200 × 106 sperm), S200 (n = 24; single AI with 200 × 106 sperm), D100 (n = 24; double AI with 100 × 106 sperm) and S100 (n = 18; single AI with 100 × 106 sperm). Ewes were inseminated within 12 to 18 h for single AI and, within 10 to 12 h and 16 to 18 h for double AI after the onset of oestrus. The onset of oestrus ranged from 28 to 76 h (54.33 ± 1.28 h). The high percentage (29.2%) of ewes showed oestrus between 51 to 60 h. The non-return rates were highest in group D200 (56.5%) and differed significantly (p < 0.05) from group S100 (11.1%). No ewes were pregnant in group S100, and the pregnancy rates among the remaining groups did not differ. The mean gestation period was 152.8 ± 0.5 days and no difference was observed among the groups. The lambing and multiple birth rates were 100% in group D200. The single and twin lambing was highest in group D100 (33.3%) and group D200 (83.3%), respectively. Only one triplet lambing and the highest lambing size (2.2 ± 0.2) was recorded in group D200. In conclusion, double AI with 200 × 106 sperm showed comparatively most practical for achieving high pregnancy rates and lambing performances in Bangladeshi ewes under field conditions.

Bisphenol‒A (BPA) is a known endocrine‒disrupting chemical used extensively to manufacture plastic bottles, canned food linings, thermal receipts, and other commonly used items. BPA is capable of inducing chromosomal alterations in germ cell line, thereby produced transgenerational effects on brain function, social recognition, reproductive diseases, sperm quality, gene expression, and obesity. Here, we aimed to investigate the transgenerational effects of BPA on murine male fertility. Six-week-old male mice (F0) were gavaged with corn oil (control), two different doses of BPA (5 mg, and 50 mg·kg bw-1·day-1),andethinylestradiol(EE,0.4mg·kg bw-1·day-1), dailyfor6weeks. Treated male mice were mated with wild‒type female and sibling pairs were bred up to the third generation (F3) in a similar manner with no further BPA exposure. Testes and spermatozoa were collected from 14-week-old males of all generation (F0 to F3) to evaluate testis weight, sperm function, and fertility. We found that high concentration of BPA significantly increased testicular weight in F2. Although the sperm viability, capacitation status, and intracellular ROS levels were not affected by BPA, however, sperm count, motility, hyperactivated motility, and intracellular ATP levels were significantly altered by BPA, dose dependently. In majority of the cases the effects were prominent in F2 followed by F1 and F0, whereas the effects were diminished in F3 generation. Simultaneously, high concentration of BPA significantly decreased cleavage and blastocyst formation rate in both F1 and F2. Similar inhibitory effects on cleavage and blastocyst were also noted in F1 by low dose of BPA. Depending on these findings we conclude that BPA decreases the fertility potential of exposed males and has an adverse impact on sperm function and fertility in subsequent generations.

Sperm adhesion molecule 1 (SPAM1) and Hyaluronidase 5 (HYAL5) has been well-known as assistants for sperm penetrate through the cumulus mass surrounding the ovulated eggs. However, so far their role in mammalian fertilization remain elusive, because mouse sperm lacking SPAM1 or HYAL5 were still capable of penetrating the cumulus mass despite a delayed dispersal of cumulus mass. Those data collectively demonstrated that SPAM1 or HYAL5 deficiency alone was not sufficient to cause male infertility in mice. In the present study, SPAM1 and HYAL5-simultaneous deficient male mice model was generated. Because of inhibition in sperm hyaluronidases, SPAM1 and HYAL5-deficient male mice produced significantly smaller numbers of offspring than hetero type and wild type mice. Hyaluronic acid degradation assay and cumulus oocyte complex dispersal assay as well as sperm motility assay using double knock out sperm and extracts had severe adverse effects on the dispersal of cumulus oocyte complex, which was the main reason for the impaired fertility of double knock out male sperm. Moreover, hyaluronic acid degradation assay using human sperm extracts revealed that sperm hyaluronidase has a principal role in sperm penetration through the cumulus oocyte complex. In conclusion, our results suggest that sperm hyaluronidase deficiency may be sufficient to cause male sterility in mammal because SPAM1 and HYAL5 deficiency sperm not impaired the sperm motility in hyaluronic acid but also cumulus oocyte complex penetration.

Jeju Black Cattle (JBC) is an indigenous species of Korea and their mass production and industrialization are required for this high quality indigenous species. For production of elite JBC zygotes, selection of high quality sperm is necessary for in vitro fertilizatioin. In this study, we compared the sperm fertility and developmental capacity of IVF embryos using various JBC sperm (Bull A, B and C). The frozen semen was thawed and confirmed sperm viability and motility. In addition, frozen-thawed sperm was used for a chlorotetracycline(CTC) staining assay and in vitro fertilization. Sperm were classified into three staining patterns. The F pattern is indicative of uncapacitated sperm, the B pattern is indicative of capacitating and capacitated sperm and the AR pattern is indicative of acrosome-reacting sperm or acrosome-reacted sperm, respectively. Several kinds of JBC sperm was inseminated in 44 ㎕ IVF drop contained 10 oocytes with sperm concentration of 1 × 106 cells/ml, and then 2 ㎕ heparin and 2 ㎕ PHE (20 μM penicillamine, 10 μM hypotaurine, 2 μM epinephrine) were added. The sperm viability and motility were higher in sperm 3 species (n=8). When we confirmed sperm capacitation, F pattern and B pattern rate were higher than AR pattern in sperm A group. After IVF, the rates of cleavage and blastocyst development were higher in sperm C group compared to other sperm group. However, the cell number of blastocyst was higher in sperm E group. These results demonstrate that the use of sperm C was effective in production of elite JBC IVF embryos. Additional experimental data are required for more accurate analysis.

본 연구는 현재 국립축산과학원 가축유전자원세터에서 보유 하고 있는 재래닭을 순수화된 품종인 것으로 판단하고, 일반 적으로 이용되고 있는 산란사료 및 사양관리 방법을 적용하여 특히, 동절기에 있어 재래닭의 정자의 보존 기간과 수정률 및 초기배자의 생존율을 각각 비교함으로써 재래닭의 생산성 향 상을 위한 기초자료를 제공하고, 나아가 표준능력을 고찰하고 자 수행하였다. 본 시험에 사용된 공시계는 39주령의 재래닭 6계통 적갈색(R, Red Brown Strain), 황갈색(Y, Yellow Brown Strain), 회갈색(G, Gray Brown Strain), 흑색(L, Black Strain), 백색(W, White Strain) 그리고 오계(O, Ogol Strain)를 대상 으로 하고 대조군으로는 3계통 즉, 외래도입종 중에서 대표적 인 다산종인 White Leghorn (F Strain), Rhode Island (C Strain) 그리고 육용종인 Cornish (H Strain)의 수정률 및 초기배자 생존율을 조사하였다. 단 한번의 인공수정 후, 3 주간 생산된 알의 수정률 확인을 한 결과, 재래닭의 경우, 6 계통간의 유의 적인 차이는 없지만 93.3 ~ 100.0비율로 인공 수정 후, 2일째 부터 수정률이 6일째 동안 최고 높음을 확인했다. 6일째까지 상대적으로 일정하게 높은 수정률을 유지하다가 7일부터 17일 째 까지 점진적으로 감소함을 확인했다. 17일 이후 생산된 알 의 경우 무정란임을 확인 할 수 있었다. 재래닭(R, Y, 그리고 O) 21일간 생산된 알의 배발생정지율의 결과, 인공수정 후, 약 4일째 생산된 알(3 ~ 6일)에서 외래도입종 3품종간의 유의적인 차이는 보이지 않았지만, 배발생정지율이 0%임을 확인하였다. 세 품종 모두 약 7일째 생산된 알부터 배발생정지율이 13.8 ~ 26.7%로 급격히 증가하고 12일부터 감소하는 것을 확 인했다. 재래닭 3 계통의 결과도 인공수정 후, 약 4 일째 생 산된 알에서 외래도입종 3 품종과 유사한 패턴을 나타내면서 배아 사망율이 6 일째까지 0%를 보였다. 금후, 체내 정자보존 기간이 수정률 및 초기배자 생존율에 미치는 영향을 좀더 엄 밀하게 조사하기 위해서는 정액 성상 및 활력 검사와 더불어 암탉의 주령에 따른 변화도 함께 보다 세부적이고 입체적인 방법의 체계적인 조사가 반드시 필요하다고 할 수 있겠다.

The main purpose of this study is to estimate the effect of adding Tea-N-Tris (TES) to the freezing buffer for miniature pig sperm. In particular, we attempted to identify the association between the MMPs expression and the fertility and viability of frozen sperm from each extender (LEY (Lactose Egg-Yolk), TLE (TES + LEY), TFGE (TES + Fructose + Glucose Egg-Yolk)). In accordance with this, Hypoosmotic Swelling Test (HOST) respond test was the lowest among sperms frozen in LEY while the highest HOST respond was observed among sperms frozen in TLE. Furthermore, we observed MMPs expression in all sperm groups, with pro-MMP showing lower expression than active MMPs. The expression of MMP-9 and MMP-2 was the highest in sperms frozen in LEY, Meanwhile, sperms from the TFGE and TLE group showed lower level of MMP-9 and MMP-2 expression in the order of TLE being the lowest. LEY group showed lower rate of blastocyst development than the TES supplement group, although the difference was not statistically significant. Meanwhile the rate of blastocyst development appeared similar when sperms from TLE and TFGE group were used for IVF. Together, these results indicate that adding Tea-N-Tris to the sperm freezing buffer only suppresses MMPs protein activation but also maximize in-vitro fertility, providing a means to improve the success rate in the in vitro manipulation of miniature pig sperm.

The objective of this study was to develop of semen transport system for cryopreservation and fertility in bull sperm. The ejaculated semen were diluted with Triladyl containing 20% egg-yolk for transportation. Diluted semen was transported by three methods that there were wrapping tissue (Tissue), sinking under 30℃ water (Water) and sinking between warm water and air (Air) methods. Semen was transported within 2 hours in 0.3℃. For this study, the freezing of diluted semen were added with Triladyl containing 20% egg-yolk. And frozen-thawed sperm were estimated with SYBR14/PI double stain for viability, FITC-PNA/PI double stain for acrosome reaction analysis and Rhodamine123 double stain for mitochondrial intact assessment. In results, live sperm (SYBR+/PI-) in Air treatment group (43.3±4.7%) was significantly (p<0.05) higher than other treatment groups (Tissue: 16.3±2.7% and Water: 27.5± 3.1%), dying sperm (SYBR+/PI+) in Air treatment group (55.6±4.7%) was significantly lower than other treatment groups (Tissue: 77.6±3.2% and Water: 67.6±3.3%) (p<0.05). Acrosome reaction in Air treatment group (0.2±0.1%) within live sperm (PI negative region) was significantly (p<0.05) lower than other treatment groups (Tissue: 0.7±0.2% and Water: 0.5±0.1%), the acrosome reaction in Air treatment group (28.6±2.8%) within all sperm also was significantly lower than other treatment groups (Tissue: 44.2±1.8% and Water: 36.2±2.0%) (p<0.05). And mitochondrial intact in Air treatment group within live (97.1±0.4%) and all (61.9±3.3%) sperm were significantly higher than other treatment groups (Tissue: 85.2±3.3%, Water: 87.8±2.9% within live sperm and Tissue: 49.28±3.7%, Water: 42.0±3.1% within all sperm) (p<0.05). Therefore, we suggest that transportation by sinking method between warm water and air was beneficial to improvement of fertility in frozen-thawed in bull semen.

The objective of this study was two folds: to investigate the relationship between paternal identification rate and sperm quality parameters such as motility and sperm chromatin structure assay after heterospermic insemination; to see if mutual complement between tests and development of useful technique to enhance the fertility in artificial insemination. In individual boar's fertilizing ability, 3 high fertility boars showed significantly high fertility (p<0.05) compared to 3 low fertility boars, but there was no difference in litter size between two groups. Sperm motility test in pooled and individual semen using computer assisted sperm analysis (CASA) revealed that no significant difference among boars. The high fertile boar showed tendency of low %Red (High red fluorescence/green+red fluorescence) in sperm chromatin structure assay (SCSA) but paternal identification rate from piglets did not differ after heterospermic insemination. The correlation coefficient between individual or pooled semen function test and farrowing rates were well correlated as follows: %Red with litter size (r= - 0.53, p=0.03); %Red with paternal identification rates (r=-0.51, p=0.03); paternal identification rates with litter size (r=0.57, p=0.02). These results indicate that sperm chromatin structure assay and sperm quality parameter test in pooled semen are useful method to predict and evaluate the fertilizing capacity after heterospermic insemination in boars.

본 연구는 체세포를 이용하여 생산된 복제 한우 수소의 번식능력을 검토하기 위해 실시하였다. 복제 한우 수소(C-38 및 C-39) 또는 일반 한우 종모우로부터 정액을 채취하여 정자의 수 및 동결 전후의 생존성 등을 살펴보았으며, 정자의 운동성 등은 computer assisted sperm analysis(CASA)를 이용하여 측정하였다. 또한, 이들의 수정 능력을 확인하기 위하여 체외수정과 인공수정을 각각 실시하였다. 정액 성상에서는 복제 수소들과 일반 종모우 간에 정액의 양, 정자의 농도 및 동결융해 후의 생존성 등에서 차이가 나타나지 않았다. CASA를 이용한 분석에서 운동성, 곡선 운동 속도(VCL), 직선 운동 속도(VSL) 및 평균 진행 속도(VAP) 등은 복제 수소의 정액이 일반 종모우의 정액에 비하여 유의적으로 높았다(p<0.05). 체외수정에 따른 수정란의 분화율 및 배반포로의 발달율은 복제 수소와 일반 종모우 간에 차이가 나타나지 않았다. 복제소 정액(C-38)을 이용하여 인공수정을 한 5두의 체세포 복제 대리모에서 암수 각각 한 두씩의 건강한 복제 후대 송아지 2두를 생산하였다. 이상의 결과를 종합하여 보면, 실험에 공시된 복제 수소 개체 간의 차이가 나타나기는 하였지만, 복제 수소는 정액 성상과 정자의 운동성 등에서 일반 종모우와 차이가 없었으며. 또한 인공수정을 통해 송아지를 생산함으로써 정상적인 번식능력이 있음을 확인하였다.

This study was conducted to compare the reproduction ability of the wild type boar and recombinant human erythropoietin (hEPO) transgenic boar semen. Ejaculated boar semen was analyzed by flow cytometry, Elisa and IVF methods. In experiment 1, flow cytometric analysis showed that the live sperm ratio of transgenic boar sperm significantly lower (P<0.05) than that of wild type boar after incubation at 20, 22, 24 and 26 hr. In experiment 2, the presence and levels of various cytokines (IL-6, IL-10 and TNF-α) to related animal reproduction in the seminal and blood plasma were examined using specific enzyme immunoassay. There was no significant difference between both groups. In experiment 3, the fertilizing capacity and developmental ability of both boar sperm were compared. The transgenic boar sperm had a significantly low capacity of penetration, sperm-zona binding, embryo development, and blastocyst formation compared to wild type sperm (P<0.05). These results suggest that transgenic boar sperm harboring hEPO gene has low sperm viability than wild type boar, and it is a reason to decrease of fertility and litter size.