쥐의 암컷에서 유리불포화지방산, 인지질, creatinine 및 prostaglandin의 혈액내 함량을 측정하여 실험 동물로써의 생리적인 기초자료를 제공하고 또한 임신일수와 분만후 경과시간에 따른 이들 성분의 변화를 관찰하여 임신과 분만에 미치는 영향에 대하여 예비정보를 제공하기 위하여 본 실험을 실시하였다. 이러한 목적을 달성하기 위하여 5마리의 임신하지 않는 쥐를 대조군으로 하고 임신한 쥐 35마리를 5마리씩 7군으로 나누어 (5개의 임신군과 2개의 분만군) 100일 동안 실험을 실시하였다. 본 실험에서 얻어진 결과를 요약하면 다음과 같다. 1) 쥐 암컷의 혈청내 유리불포화지방산 함량은 의 범위였고 각 실험군간에는 현저한 차이가 없었다. 그러나 임신21일군이 로 비교적 낮은 수준이었다. 2) 쥐 암컷의 혈청내 인지질 함량은 의 범위였고 임신21일군과 분만후 36시간군이 비교적 낮은 수준이었지만, 각 실험군 및 전체임신군과 전체분만군의 평균함량간에는 현저한 차이는 없었다. 3) 쥐 암컷의 혈청내 creatinine 함량은 의 범위였고 각 실험군간에 현저한 차이가 안정되지 않았지만, 임신21일군이 비교적 높은 수준이었다. 4) 쥐 암컷의 혈액내 prostaglandin 함량은 로써 대조군()에 비하여 임신9일(), 12일() 및 15일()군 등이 낮은 수준인데 비하여 임신18일군()부터 대조군보다 증가되기 시작하여 임신21일군()과 분만후 12시간군()까지 계속 증가하여 최고수준에 달했으며 분만후 36시간군()도 대조군에 비하여 현저하게 높은 수준이었다. 그리고 각 실험군간에는 현저한(p<0.01)차이가 안정되었고 특히 전체임신군의 평균함량이 로써 대조군에 비하여 낮았고, 전체분만군의 평균함량이 로써 대조군과 천체임신군의 평균함량보다 매우 높은 수준이었다. 5) 쥐 암컷의 혈청내 유리불포화지방산, 인지질 및 creatinine 함량은 임신일수와 분만후 경과시간에 따라 큰 영향을 받지않는 것으로 생각되지만, 임신말기와 분만후에 약간의 변화가 있었다. 6) 쥐 암컷의 혈액내 prostaglandin 함량은 분만개시에 중요한 역할을 한다고 생각된다.

This study was carried out to investigate the inte actions between recipients and embryos to compare pregnancy rates in bovine embryo transfer, such as synchrony and stage of embryos, synchrony and quality of embryos, synchrony and side of uterine horn, and preservation time and stage of embryos. Fifty-two embryos were transferred by surgically to 42 Holstein heifers, 3 Holstein cows and 7 Korean native heifers from Feb., 1985 to June, 1986. The results were as follows: 1. In the trial of interactions between synchrony and embryo stage, recipients synchronired from- hours to + 12 hours in synchrony and embryos from morulac stage to advanced blastoctyst stages showed reason able pregnancy rate. 2. Excellant (A) and good (B) grade embryos showed good pregnancy rate, 81.8% and 73.3% respectively, but fair (C) grade embryos showed poor, 25% only at the same boundary of recipient synchrony. 3. More recipients had corpus luteum on the right ovary than the left, and also had better pregnancy rate (26/32 vs 13/20,81.3% vs 65%). 4. A good pregnancy rates (over 60%) were obtained with the embryo transferred 17hrs after flush at room temperature or field condition in culture medium.

Most of the commercial devices for vitrification are directly immersed into the warming solution (WS) for increasing of warming rate. However, the previous modified cut standard straw (MCS) which has reported is difficult to immerse into the WS. The aim of this study was to investigate whether the long cut straw (LCS) could be useful as a stable tool for vitrified-warmed human blastocysts. A total of 138 vitrified-warmed cycles were performed between November 2013 and November 2014 (exclusion criteria: women ≥38 years old, poor responder, surgical retrieval sperm, and severe male factor). The artificial shrinkage was conducted using 29-gauge needles. Ethylene glycol and dimethyl sulfoxide (7.5% and 15% (v/v)) were used as cryoprotectants. Freezing and warming were conducted using the LCS tool. The cap of LCS was removed using the forceps in the liquid nitrogen (LN2) and then directly immersed into the first WS for 1 min at 37℃ (1 M sucrose). Only re-expanded blastocysts were transferred after it was cultured in sequential media for 18-20 h. A total of 294 blastocysts were warmed, and all were recovered (100%). Two hundred eighty-five embryos were survived (96.9%). The vitrifiedwarmed blastocysts of all patients were transferred without any cancellation. We were able to achieve a reasonable implantation (24.2%), following by clinical pregnancy (36.2%), which then continued to ongoing pregnancy (36.2%), and live birth (31.2%). Using LCS is achieved the acceptable rates of survival, pregnancy and live birth. Therefore, the LCS could be considered as a stable and simple tool for human embryo vitrificaton.

The majority of early conceptus mortality in pregnancy occurs during the peri-implantation stage, suggesting that this period is important for conceptus viability and the establishment of pregnancy. Successful establishment of pregnancy in all mammalian species depends on the orchestrated molecular events that transpire at the conceptus-uterine interface during the peri-implantation phase. This maternal-conceptus interaction is especially crucial in pigs because in them non-invasive epitheliochorial placentation occurs, in which the pre-implantation phase is prolonged. During the pre-implantation period, conceptus survival and the establishment of pregnancy are known to depend on the developing conceptus receiving an adequate supply of histotroph, which contains a wide range of nutrients and growth factors. Evidence links epidermal growth factor (EGF), insulin- like growth factor-I (IGF-I), vascular endothelial growth factor (VEGF), and colony-stimulating factor 2 (CSF2) to embryogenesis or implantation in various mammalian species; however, in the case of pig, little is known about such functions of these growth factors, especially their regulatory mechanisms at the maternal-conceptus interface. Therefore, the objectives of this study were to determine: 1) the temporal and cell-specific expression of EGF, IGF-I, VEGF, and CSF2 signaling systems in the porcine endometrium during the estrous cycle and early pregnancy; 2) the potential intracellular signaling pathways responsible for the activities of these four factors in primary porcine trophectoderm (pTr) cells; and 3) the changes in cellular activities induced by these promising factors. First, the functional effect and cellular signaling cascades in pTr cells induced by EGF, which exhibits potential growth-promoting activities on the conceptus and endometrium, were investigated. EGFR mRNA and protein were abundant in endometrial luminal epithelia (LE) and glandular epithelia (GE), stratum compactum stroma, and conceptus trophectoderm on Days 13-14 of pregnancy, but not in any other cells of the uterus. EGF treatment of pTr cells increased the abundance of phosphorylated (p)-AKT1, p-ERK1/2 MAPK and p-P90RSK in the nucleus and/or cytoplasm when compared with the levels in control cells. Furthermore, EGF-stimulated phosphorylation of AKT1 and ERK1/2 MAPK were inhibited in pTr cells transfected with an EGFR siRNA, and compared with control siRNA- transfected pTr cells, the EGFR siRNA-transfected pTr cells exhibited an increase in the expression of gene encoding interferon (IFN)-δ and transforming growth factor (TGF) β-1; by contrast, no effect was detected on the expression of the gene encoding IFN-γ. Moreover, EGF stimulated the proliferation and migration of pTr cells, but these stimulatory effects were blocked by pharmacological inhibitors such as SB203580 (a p38 inhibitor), U0126 (a MAPK inhibitor), rapamycin (an MTOR inhibitor), and LY294002 (a PI3K inhibitor). Second, IGF-I was examined. IGF-1 is another promising growth factor that is known to play key roles in reproductive processes; however, little is known about IGF-I-induced functional effects and regulatory mechanisms during peri-implantation in pigs. In this study, endometrial type I IGF receptor (IGF-IR) mRNA was determined to increase substantially during early pregnancy relative to the level during the estrous cycle, and the mRNAs of both IGF-I and IGF-IR were abundant in endometrial LE and GE, stroma and conceptus trophectoderm on Day 12 of pregnancy. Moreover, IGF-I treatment potently increased the amounts of p-AKT1 and, ERK1/2 MAPK in the nucleus and cytoplasm and of RPS6 in the cytosol when compared with the amounts in untreated pTr cells, and IGF-I-induced activation of AKT1 and ERK1/2 was blocked by LY294002. Furthermore, IGF-I strongly stimulated both the proliferation and the migration of pTr cells, but these effects were inhibited by SB203580, U0126, rapamycin and LY294002. Third, this study focused on VEGF, which was identified as a potential mediator of the fetal-maternal dialog that regulates the development of the peri-implantation porcine conceptus. In addition to its known angiogenic effects, VEGF has been suggested to play roles in the development of the early embryo, but VEGF-induced effects on the peri-implantation conceptus remain unknown. Results of this study revealed that endometrial VEGF, VEGF receptor (VEGFR)-1, and VEGFR-2 mRNA levels in endometrial LE and GE, endothelial blood vessels, and scattered cells in the stroma were more abundant during the peri-implantation period of pregnancy than during the estrous cycle. Moreover, VEGF treatment of pTr cells increased the abundance of p-AKT1, p-ERK1/2, p-p70RSK, p-RPS6 and p-4EBP1, and the addition of LY294002 suppressed VEGF-induced phosphorylation of ERK1/2 and AKT1. Furthermore, VEGF potently stimulated both the proliferation and the migration of pTr cells, but these effects were inhibited in the presence of SB203580, U0126, rapamycin and LY294002. The fourth promising cytokine studied was CSF2, which is also known as granulocyte-macrophage colony-stimulating factor (GM-CSF). CSF2 plays a role in facilitating mammalian early embryonic development. In this study, endometrial CSF2 mRNA expression was determined to be increased during the peri-implantation period relative to the mRNA level during the estrous cycle. In pTr cells, CSF2 significantly induced the activation of AKT1, ERK1/2, MTOR, p70RSK, and RPS6, but not of STAT3, and the addition of LY294002 abolished CSF2-induced increases in p-ERK1/2, p-MTOR, and p-AKT1 levels. Furthermore, CSF2 strongly stimulated pTr cell proliferation, an effect that was blocked by U0126, rapamycin and LY294002. Collectively, these results provide new insights into the potential mediators that regulate the development of the peri-implantation conceptus at the fetal-maternal interface. These results indicate that endometrial- and/or conceptus derived EGF, IGF-I, VEGF, and CSF2 critically affect the growth and development of porcine trophectoderm cells, and that these stimulatory effects are coordinately regulated by multiple cellular signaling cascades including the PI3K-AKT and ERK1/2 MAPK pathways during early pregnancy in pigs.

The implantation process in pigs is initiated when the conceptus begins secretion of estrogen, the signal for maternal recognition of pregnancy, and cytokines including interleukin-1β(IL1B), interferon delta (IFND) and interferon gamma (IFNG). Our previous study showed that IFNG receptors, IFNGR1 and IFNGR2, were expressed in the uterine endometrium during the estrous cycle and early pregnancy. However, the molecular and cellular mechanism of IFNG in the uterine endometrium in pigs is poorly understood. To determine the role of IFNG on the uterine endometrium during the implantation period, we took advantage of RNA-Seq analysis using explant tissues treated with IFNG in the presence of estrogen and progesterone, and found that many genes including CXCL9, CXCL10, CXCL11, IDO1, IL15, IL15RA, TNFSF10 (TRAIL), and WARS were up-regulated by IFNG. Additional analysis in the uterine endometrial tissues from day (D) 12 and D15 of the estrous cycle and from D12, D15, D30, D60, D90 and D114 of pregnancy determined the expression of these IFNG-regulated genes in pigs by quantitative real-time PCR Results showed that expression of CXCL9, CXCL10, and IDO1 dramatically increased on D15 of pregnancy, and expression of CXCL11 and TNFSF10 was high during mid- to term pregnancy. These results indicate that IFNG regulates immune-associated genes in the uterine endometrium in a stage-specific fashion during pregnancy, and may play a critical role to support the establishment and maintenance of pregnancy at the fetomaternal interface in pigs.

Na+/K+-ATPase, an energy-transducing ion pump, is responsible for maintenance of relatively high concentrations of potassium ions but low concentrations of sodium ions in the cell by transport of these ions across the plasma membrane and participates in transport of various nutrients including glucose, amino acids, and ions. In addition, Na+/K+-ATPase is also involved in regulation of intracellular calcium ion concentration by coupling with Na+/Ca+ exchanger expressed at the maternal-fetal interface in pigs. Na+/K+-ATPase consists of α, β, and FXYD subunits, but only α and β subunits are required for primary functions. FXYD subunit is an auxiliary protein for αβ complex of Na+/K+-ATPase. However, it has not been determined that subunits of Na+/K+-ATPase are expressed in the uterine endometrium during the estrous cycle and pregnancy in pigs. In this study, we determined expression of alpha (ATP1A1-4), beta (ATP1B1-3), and FXYD (FXYD1-7) subunits of Na+/K+-ATPase in the uterine endometrium during the estrous cycle and pregnancy in pigs. Real-time RT-PCR analysis showed that all alpha, beta, and FXYD subunits, except ATP1A3, were expressed in the uterine endometrium during the estrous cycle and pregnancy in a pregnancy status- and stage-specific fashion. In situ hybridization analysis exhibited that ATP1A1, ATP1A4, and ATP1B1 were localized to luminal (LE) and glandular epithelium (GE) during the estrous cycle and early pregnancy, and during mid to term pregnancy. ATP1A1 mRNA was localized to LE, GE, and areolae of the chorioallantois, especially at high levels to LE in areolae regions. ATP1B3 mRNA was detected only in LE during the estrous cycle and pregnancy with highest levels on day (D) 12 of pregnancy. Transcripts of all subtypes of FXYD subunit were primarily localized to LE and GE during the estrous cycle and early pregnancy and to chorionic membrane (CM) during mid to term pregnancy. RT-PCR analysis showed that all subtypes of Na+/K+-ATPase subunits, except ATP1A2, ATP1A3, and ATP1B2 mRNAs, were expressed in conceptuses on D12 and D15 of pregnancy. These results indicate that Na+/K+-ATPase subunits are expressed in the uterine endometrium and conceptuses during the estrous cycle and pregnancy in a pregnancy status- and stage-specific manner. These suggest that Na+/K+-ATPase subunits may be involved in the establishment and maintenance of pregnancy by coordinate regulation of absorption and secretion of nutrients such as glucose, amino acids, and ions at the maternal-fetal interface in pigs.

Bisphenol A(BPA)는 약한 estrogen 활성도를 보이는 화학물질로서 착상 전 배아나 태아에 영향을 미쳐, 출생 후 그들의 신체발달에 변화를 초래한다고 알려져 있다. 본 연구는 인체에서 임신 중기에 양수 내 BPA 농도를 측정하였으며, 측정된 농도에 따라 임신결과에 영향을 미치는지 여부를 알아보고자 하였다. 임신 15주에서 20주 사이에 유전학적 적응증으로 양수천자 검사를 시행 받았던 120명의 임신부 양수를 연구대상으로 ELISA 방법으로

Apoptosis of the cell is one of the key steps in tissue remodeling and functional differentiation. At the time of implantation there are dramatically remodeling and functional differentiations of uterine endometrium. Decidualization is the process by which the uterine stromal cells proliferate and differentiation into morphologically and functionally distinct decidual cells. Bax is one of the key molecules in apoptotic process during that time. It is known that Bax expression have characters of time specificity and tissue specificity but the role of implantation is not largely unknown. The aim of this study was to examine the embryo implantation and decidualization reaction in order to understand the role of pro-apoptotic Bax gene as a major regulator of apoptosis of decidual cells. Some of Bax knockout mice have coat pigmentation defects involving white color. The number of blue bands after embryo transfer (ET) was not different between the control and Bax-knockout (Bax-KO) black coat (BC) and white coat (WC). Decidua were normally differentiated in both control and Bax-KO BC, but deciuda were not detected in Bax-KO WC at 96 hr post ET. Moreover, embryos of Bax-KO WC stayed in dormant stage on the embryonic day 7.5 and day 9.5. These results suggest that Bax and its related unknown genes may be a key molecule in implantation and uterine decidual reaction.

수정, 착상부터 임신 후반기까지 영장류의 생식활동은 모체와 태아간 면역계의 관용과 거부의 균형에 의존하게 되며, 이런 면역반응은 부성의 major histocompatibility complex (MHC)를 가진 배아 또는 태아에 의한 면역계의 자극을 모체가 인지함으로써 시작된다. 태아에 대한 모체의 면역반응은 “면역관용, immune tolerance”이라는 자궁 특유의 면역학적 특징을 보인다. 1950년대 Medawar 등은 태아가 모체 내에서 면역계의 공격을 이기고 안전하게 성장할 수 있는 기전으로 i) 모체 면역세포가 태아항원에 대한 무반응 (anergy) 또는 면역관용을 획득한다는 가설, ii) 태아와 모체간 해부학적 장벽에 의해 모체 면역세포가 태아세포에 접근할 수 없다는 가설, 그리고 iii) 태아세포 스스로 동종항체의 발현을 억제한다는 가설 등을 제기하였다. 하지만 Medawar의 가설은 초창기 생식면역학의 기본 개념을 제시하였지만 태아항원과 모체 면역계 사이에 이루어지는 면역관용 현상을 설명하는데는 부족함이 많았다. 이후, 자연살해세포 (natural killer, NK cell), 자연살해 T 세포 (natural killer T, NKT cell), 면역조절 T 세포 (regulatory T, Treg cell), 단핵세포 (monocyte), 수지상세포 (dendritic cell), 대식 세포 (Macrophage) 등 다양한 종류의 모체 면역세포들이 면역관용에 관여한다는 연구들이 보고되었다. 이들 면역세포들은 자궁 내의 태아와 모체가 접촉하는 “태아-모체 접촉면, feto-maternal interface”에 decidual associated lymphoid tissue (DALT)라는 임신 특이의 조직학적 구조물 내에 존재하며, 태아에 대한 모체의 면역관용을 획득하는데 중요한 역할을 하고, 착상 및 임신 유지에 수반되는 혈관생성 및 영양막 (trophoblast)의 발달에 필수적인 무균성 염증반응 (sterile inflammation)을 유발하는 것으로 알려졌다. DALT에서 모체의 면역세포들과 여러 전달물질간의 복잡하고 다양한 연결망의 형성에 의한 적절한 염증반응은 배아의 착상 및 임신의 유지에 필수적인 혈관생성 및 영양막의 발달에 중요한 역할을 하는 반면 적절하지 않거나 과도하게 오랜기간 지속되는 염증반응은 급성 혹은만성 이식거부 반응과 유사한 작용을 일으켜 태반의 성장, 태아의 성장 및 발달에 심각한 장애를 초래하며, 착상부전(repeated implantation failure), 습관성 유산 (recurrent spontaneous abortion), 임신 자간증 (preeclampsia) 등 그 병인이 유사한 생식능력장애 (defect of reproductive performance)의 원인이 될 수 있다. 인간 탈락막 또는 자궁내막에 존재하는 면역세포는 극소수의 백혈구만 존재하며, 주로 세가지 아형의 면역세포 즉, T 림프구, 대식세포, 자궁 내 자연살해세포 등이 주를 이루고, B 림프구가 거의 없는 점 등에서 말초혈액의 면역세포의 구성과 많은 차이가 있다. 그간의 연구결과를 종합해 보면 착상과 임신의 유지를 위해서는 태아의 동종항원에 대한 모체의 면역반응의 균형이 필수적이며, 이러한 균형에 문제가 생겨 과도한 면역반응이 유도될 경우, 태반의 성장에 심각한 장애를 초래하여 착상부전 (recurrent implantation failure, RIF)이나 반복적 유산 (recurrent spontaneous abortion, RSA), 전자간증과 같은 생식부전 (reproductive failure)의 원인이 될 수 있다. 임신 중 태아에 대한 모체의 면역에 관여하는 세포들 중 NK cell의 착상과 임신을 유지하는 과정에서의 역할에 대한 연구가 최근 비교적 활발히 이루어지고 있다. 특히, NK cell의 적절한 면역반응에 대한 조절을 실패는 습관성 유산, 불임 (infertility), 그리고 전자간증 (pre-eclampsia) 같은 병리학적 기전들과 관련이 있다고 보고되고 있으며, 특히, 근래에 습관성 유산의 원인과 관련하여 NK cell에 대한 많은 연구가 이루어지고 있다. 말초혈액에서 NK cell(periopheral blood NK cell, pbNK cell)은 대부분이 CD56, CD16 세포 표면 항원을 발현하고 강한 세포 독성 (cytotoxicity)을 가지는 반면, CD56 세포 표면 항원은 강하게 발현하지만 CD16 세포 표면 항원은 발현하지 않는 자궁 내 NK cell (decidual NK cell, dNK)은 세포독성은 약하고 다양한 종류의 cytokine을 분비하는 특징을 가지는 것으로 보고되고 있다. 여러 문헌에 의하면, 정상 임신 초기에는 pbNK cell의 활성도와 분획은 감소하는 반면, 습관성 유산 환자에서는 증가되어 있고, 임신을 확인하기 전에 측정한 경우 역시 습관성 유산의 경력이 있는 여성에서 pbNK cell의 비율이 높게 나타나 pbNK cell의 세포용해 활성도 또는 분획의 증가는 연속되는 임신에서 습관성 유산의 위험성을 증가시키는 원인으로 보고되었으며, 그 결과는 NK cell의 면역작용에 대한 성공적인 조절이 임신의 성공 및 유지에 중요한 요소라는 결론이 제시되었다. 한편, 유산이 진단된 유산산물의 탈락막 내에서 CD56+CD16－ 형태의 dNK cell이 높게 검출되어 dNK cell의 작용부위가 착상부위임을 보고하였으며, 결국 습관성 유산 및 착상 부전 등 NK cell에 의한 생식 부전의 진단에는 자궁 내의 NK cell의 세포용해 활성도와 분획의 분석이 정확한 진단에 도움이 될 것으로 여겨지지만, 검체 채취의 윤리성과 검사방법의 어려움으로 연구 수행에 많은 제한이 있다. 본 강의에서는 임신의 성공과 유지에 관련 하여 NK cell의 역할 및 착상부전, 습관성 유산을 예측하는데, NK cell의 진단적 기능에 대해 문헌고찰 및 저자의 연구결과를 중심으로 논의하고자 한다.

Pregnancy-associated plasma protein-A (PAPP-A) is a 200 kDa metalloprotease identified as an IGFBP-4 protease and likely an important regulator of IGF bioavailability. PAPPA mRNA is detected in bovine granulosa and theca cells and the PAPP-A protein is also found in follicular fluid. Proteolytic activity supposed to be due to the PAPP-A in bovine follicular fluid is induced by follicle stimulating hormone (FSH) treatment and PAPP-A-like activity appears concomitantly with increased E2 during follicular development. However, the effects of FSH, E2 and progesterone on the expression of PAPP-A in bovine corpus luteum of pregnancy have been evaluated in a limited number of studies. This study was performed to expression of PAPP-A and progesterone during early pregnancy in bovine corpus luteum. To determine the function of PAPP-A gene during early pregnancy, we collected the bovine pregnancy corpus luteum samples on 30, 60 and 90 day of pregnancy. The mRNA expression of PAPP-A, progesterone-receptor, VEGF and IGFBP4 gene was conducted by real-time PCR. And proteins expression of PAPP-A and progesterone antibody was detected by Immunohistochemistry and ELISA. The VEGF and PAPP-A mRNA expression was progressively increased on day 90 in the pregnancy corpus luteum. The mRNA expression of progesterone-receptor, IGFBP4 in the corpus luteum progressively was increased from 30 to 60 day, but decreased on 90 day of pregnancy. The proteins expressions of progesterone and PAPP-A were similar pattern in mRNA expression. Our results indicate that the IGFBP4 protease role of PAPP-A increases in response to pregnancy 90 day corpus luteum and suggest that progesterone is an connected for the expression of PAPP-A genes in luteal cells. Therefore, we suggest that PAPP-A stimulated with progesterone play a crucial role for IGF-I system in bovine early pregnancy. And could be used to predict the condition of normal pregnancy.

The objective of this study was to evaluate the effect of GnRH or estradiol in a CIDR-based timed Al (TAI) protocol on follicular turnover, synchronized ovulation and pregnancy rates in Holstein cows. Cows were treated at random stages of the estrus cycle with an insertion of an intravigal progesterone (1.9 g) device (CIDR, Day 0) and either no other treatment (control group; n=10), injection of 100 ug fertirelin acetate (GnRH group; n=10) or 4 mg estradiol benzoate (estradiol group; n=10). Seven days later devices were removed and an injection of 25 mg PGF2a was administered. On Day 9, 100 ug GnRH was administered. Cows received a fixed-time insemination 16 h after injection of the GnRH.