open access

https://doi.org/10.14943/doctoral.k15209

https://hdl.handle.net/2115/90381

乳由来オステオポンチンが乳用リピートブリーダー牛における子宮内膜上皮成長因子発現の正常化と受胎性回復に及ぼす効果

英語

2022-09-26

Hokkaido University

44 p.

　　　 Endometrial EGF concentrations show a cyclic change with two peaks on days 2-4 and 13-14 during the estrous cycle. These peaks are absent or lowered in approximately 70% of Holstein lactating repeat breeder cows. The altered profile has been linked to reduced fertility. A form of OPN in bull SP normalized the EGF profile and restored fertility in the repeat breeder cows. The purpose of this study was to examine effects of OPN from milk, a rich source of this protein, on the normalization of the endometrial EGF profile and restoration of fertility in repeat breeder cows. The study is composed of two parts, the first part (Chapter 1) examined effects of mOPN to normalize the endometrial EGF profile and restore fertility in repeat breeder cows. The second part (Chapter 2) examined the feasibility of the treatment protocol using mOPN of the repeat breeder cows own milk samples. OPN contents in milk samples of apparently normal cows and repeat breeder cows at different lactation stages were examined. Then, 1 mg of mOPN was prepared using milk samples of individual repeat breeder cows and examined its effect on the EGF profile and fertility in repeat breeder cows. In Chapter 1, effects of mOPN to normalize the endometrial EGF profile and restore fertility were examined in repeat breeder cows. OPN was purified from 1 L of bovine milk by one-step of anion-exchange column chromatography. The OPN fractions showed three major protein bands of 61, 37 and 31 kDa on SDS-PAGE. All three bands were identified as OPN by Western blotting, and their tryptic peptide masses matched approximately 50, 40 and 10%, respectively, to bovine OPN amino acid sequences by peptide mass finger printing analysis. Sum of the three detected bands accounted for approximately 85% of total protein contents of the OPN preparation. OPN was obtained between 6.3 and 23.4 mg from 1 L of bovine milk. A lyophilized OPN preparation containing 1.3 mg of mOPN, 0.5 mL of SP (positive control), and PBS (negative control) were infused immediately after AI into the vagina of repeat breeder cows with an altered EGF profile. The normalization rate of EGF profile (56.1%, n = 171) was similar to that of SP (58.1%, n = 62) and higher than that of PBS (23.8%, n = 84) (P < 0.05). Similarly, the conception rate after the infusion of mOPN (43.5%, n = 46) was similar to that of SP (40.0%, n = 50) and higher than that of PBS (22.2%, n = 45) (P < 0.05). The present results confirmed the effect of seminal OPN on the normalization of EGF profile. However, using OPN prepared from raw milk for the treatment of many other cows carries the risk of spreading the infection. Thus, an alternative strategy to use mOPN for the treatment of repeat breeder cows is needed. In Chapter 2, therefore, I have examined the feasibility of treatment protocol for the repeat breeder cow with mOPN prepared from her own milk to avoid the risk of disease transmission through the treatment. Firstly, OPN content was measured in milk samples from apparently normal cows and repeat breeder cows with different lactation stages: colostrum (within two days after parturition, n = 9), early (5 to 100 days postpartum, n = 13), mid (101 to 200 days postpartum, n = 5) and late (201 to 300 days postpartum, n = 5) lactation stages to estimate milk sample volume that is necessary to obtain OPN enough (1 mg) for treatment. OPN contents were higher in the colostrum than the other lactation stages (P < 0.05). OPN contents of the other stages were at similar levels. The contents of mOPN in repeat breeder cows were similar to those of the control cows. Milk OPN of 1.0 to 3.3 mg was obtained from 100 mL of milk. Three major bands of 61, 37 and 31 kDa were detected on SDS-PAGE and all three bands were identified as OPN by Western blotting. Within control cows, the proportion of the three bands differed between the lactation stages (P < 0.05). However, proportion of band I and II were similar and higher than band III at the early and mid stages while the proportion of three bands were similar in the milk sample at the late stage. When the proportion of the three bands were compared between the control and repeat breeder cows, the proportion of the bands differed by the type of cows but not by the lactation stages. Then, 1 mg of OPN prepared from milk samples of the normal and individual repeat breeder cows was used for the treatment. Infusions of the mOPN of normal and repeat breeder cows normalized the endometrial EGF profile at similar rates of 63.3% (n = 30) and 60.0% (n = 30), respectively, and the normalization rates were higher than that of the PBS group. However, effects of mOPN from repeat breeder cows own milk remained to be confirmed since the conception rate were at an intermediate level of the PBS and mOPN of the normal cow groups. The present study indicated that use of mOPN from individual repeat breeder cows could be an effective treatment option to enabling normalization of the endometrial EGF profile and avoiding the risk of spreading infection. However, the effect of mOPN from repeat breeder cows on the restoration of fertility remained to be confirmed in a study with an increased number of animals. Nevertheless, high content of OPN and its clear effect on the normalization of the EGF profile make the mOPN a promising candidate source of OPN preparation for the treatment of repeat breeder cows with an altered endometrial EGF profile.

2022-09-26

甲第15209号

博士(獣医学)

(主査) 教授 坪田 敏男, 教授 片桐 成二, 教授 松井 基純, 助教 栁川 洋二郎

獣医学院(獣医学専攻)

博士論文

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