應用選性精液與羊水幹細胞治療對於提高荷蘭牛繁殖效率之研究

Abstract

選性精液現已廣泛應用於乳牛的繁殖計畫中，其具有提高產業競爭力與加快遺傳改良速度之優點；選性精液在流式細胞儀分選過程中，往往有可能會導致精子結構的損傷而降低其活力，荷蘭女牛使用選性精液會顯著降低懷孕率，儘管懷孕率偏低，但是近年來酪農業具有趨向於使用選性精液配種之趨勢，其中部分的原因是由於犢牛雌性比率約達到90%。早期研究結果顯示，若有明顯發情之女牛應該有排卵前濾泡 (直徑10 ~ 20 mm)；因此藉由超音波監測排卵前濾泡後，再進行人工授精應該可以改善使用選性精液之懷孕率。另一方面，為了提高乳牛乳產量亦對牛隻也造成了一些負面影響，而低生育力的主要原因是繁殖障礙， 荷蘭牛因繁殖淘汰主要的原因是卵巢衰竭、囊腫及粘黏，其中卵巢衰竭所佔的比例最高；近年來研究結果顯示，羊水是一個含有豐富來源多能間葉幹細胞的組織，羊水幹細胞可以成為一種新的成體幹細胞來源，有低免疫原性與容易獲得之特性 ，因此羊水幹細胞應具有臨床上運用於治療卵巢衰竭之潛能。(亞)熱帶地區以選性精液進行人工授精，在一年中以最熱之月份其懷孕率最低者，其中女牛懷孕率介於35 ~ 40%。試驗中挑選具排卵前濾泡之女牛，將選性精液置放於子宮體或單側子宮角中端，從而比較兩者間之懷孕率。試驗結果證明，台灣亞熱帶地區，熱季使用排卵前濾泡之女牛進而配種者，其懷孕率可以達到70%以上之譜，授精後大約40天單側子宮角中端人工授精組的總體懷孕率（71%）高於子宮體人工授精組 （42%）(P > 0.05)。此外，探討牛之羊水幹細胞治療荷蘭牛雙側卵巢衰竭之潛能，牛之羊水幹細胞採集自屠宰場懷孕期3 ~ 4個月之荷蘭牛，細胞採集後進行體外培養。牛之羊水幹細胞從第三代後開始表現出成纖維母細胞樣型態。羊水幹細胞經由RT-PCR檢測結果顯示：β-integrin, CD44, CD73, CD106與Oct-4等，分別均表現呈陽性，而CD34與CD45則分別均表現呈陰性；經適當體外誘導後，則能夠被分化成中胚層譜系。本試驗幹細胞注射的荷蘭牛選用16頭 2 ~ 3產之泌乳牛，此等牛隻分別係處於不發情與卵巢無動情週期變化之狀況者，藉由超音波掃描結果，分別確定其雙側卵巢功能係處於衰竭狀態者；經逢機選取其中8 頭做為偽注射組牛隻，另8頭則做為羊水幹細胞治療組之牛隻。試驗共進行十五週，在第1 ~ 6週期間監測牛隻之發情，若牛隻未發情則在第7週施行偽注射或牛之羊水幹細胞注射，並從第8 ~ 15週持續監測牛隻之發情情形，每週除採集血清提供進行其助孕素濃度之分析外，並且每週藉由超音波掃描紀錄觀察卵巢週期之影像變化。每頭牛隻配有發情計步器以利觀察發情及配種紀錄，各牛隻並於授精後大約40天分別藉由超音波掃描診斷其懷孕情形。在試驗期第1 ~ 9週助孕素濃度於偽注射組與牛之羊水幹細胞治療組之間並無顯著差異，但於試驗期第10週牛之羊水幹細胞治療組助孕素濃度顯著高於偽注射組 (P < 0.01)，並且於試驗期第11 ~ 15週牛之羊水幹細胞治療組助孕素濃度極顯著高於偽注射組 (P < 0.001)；此結果表示，經牛之羊水幹細胞治療後之牛隻，可藉由發情配種進而成功懷孕，因此提高血清中助孕素之濃度。在超音波診斷方面，在試驗期間偽注射組之牛隻其卵巢並無濾泡或功能性組織存在；惟牛之羊水幹細胞治療組牛隻於注射細胞後，則可以見及排卵前濾泡，並於授精後大約40天藉由超音波診斷時可以看見胚胎之存在。此外就偽注射組與牛之羊水幹細胞治療組兩者牛隻之發情比率而言，分別為0 (0/8)與50% (4/8)，成功懷孕之比率則分別為0 (0/8)與25% (2/8)。綜上所述，使用選性精液進行人工授精時，應選擇具有排卵前濾泡之女牛進行單側子宮角中端人工授精，以提高(亞)熱帶地區女牛使用選性精液人工授精之懷孕率。此外，試驗結果且證明羊水幹細胞不僅有治療乳牛卵巢衰竭之作用，且具有恢復母牛生育之能力。

Sex-sorted (Sexed) semen is now widely used in cattle breeding to enhance industrial competitiveness and accelerate the rate of genetic improvement. In the process of flow cytometry sorting, this processes often lead to damage to sperm structure, decreasing its viability. Holstein heifer use Sexed semen will significantly reduce the pregnancy rate. Despite the relatively low pregnancy outcome, the dairy industry is witnessing a trend of switching to Sexed semen artificial insemination (AI), this is in part due to higher than 90% female rates in calves. Early studies showed that female cattle with estrus cycle should have pre-ovulatory follicles (10 ~ 20 mm) if they possess obvious estrous cycle conditions. Therefore, when sexed semen was used to monitor the pre-ovulatory follicles by AI by ultrasound should improve pregnancy rates. On the other hand, the development of economic guidelines to increase the milk supply has caused some negative effects, the primary causes of low fertility are reproductive disorders, the proportion of Holstein cows culled due to ovarian dystrophy, cysts, and adhesions, ovarian dystrophy accounting for the majority. In previous studies show, amniotic fluid is a rich source of multipotent mesenchymal stem cells (MSCs). Amniotic fluid stem cells (AFSCs) have become a new source of stem cells, they have low immunogenicity and are easily harvested. For this reason, they may be useful the potential to restore ovarian function in clinical. In subtropical (ST) regions, the pregnancy rates by Sexed AI are reportedly the lowest in the hottest months yearly, with less than 35 ~ 40% in heifers. We compared Sexed AI by depositing the semen into the uterine body (UB-AI) versus the unilateral uterine horn middle-end (UUH-AI) of pre-ovulation heifers. We demonstrated that high pregnancy rates (>70%) by Sexed AI can be achieved when heifers with pre-ovulation follicles are used in a ST region.The pregnancy rate were higher in the UUH-AI (71%) group than in the UB-AI (42%) group (P > 0.05), examined on approximately 40 days post-Sexed AI. The therapeutic potential of AFSCs to treat bilateral ovarian dystrophy in dairy cows. The utility of BAFSCs injection into ovary to treat bilateral ovarian dystrophy (BOD) in Holstein dairy cows. BAFSCs were collected at slaughter from Holstein cows during the third or fourth month of pregnancy and cultured in vitro. The BAFSCs began to show a fibroblast-like morphology. Using reverse transcriptase-polymerase chain reaction analysis of gene expression that they were positive for β-integrin, CD44, CD73, CD106, and Oct-4 and negative for CD34 and CD45. After induction, the cells differentiated into mesodermal lineages. BOD was confirmed by ultrasonography in 16 lactating cows. The subsequent experiment lasted 15 weeks. Estrus cycles of cows were monitored during the weeks 1 ~ 6. The sham and / or bovine amniotic fluid stem cells (BAFSCs) injection was administered during the 7th week, and we monitored the cows’ estrus from weeks 8 ~ 15. Serum was collected weekly to analyze progesterone concentrations, and weekly ultrasonography recorded ovarian changes. Each cow was equipped with an automatic heat detection system to facilitate estrus observation and breeding records. The progesterone concentration of 2 cows in the treatment group (25%) significantly increased during weeks 10 ~ 15. The treatment group demonstrated mature follicles after BAFSCs injection. Estrus rates in the control and treatment groups were 0% (0/8) and 50% (4/8), respectively; pregnancy rates were 0% (0/8) and 25% (2/8), respectively. Calves were successfully delivered of pregnancy. Taken together, the study that Holstein heifer breeders perform UUH-AI using Sexed semen for heifers with pre-ovulation follicles in order to achieve satisfactory pregnancy outcome in ST regions. In addition, our findings BAFSCs can restore fertility for the treatment of BOD in Holstein dairy cows.