Treatments 21 days Postpartum
6.5 Summary
Despite CL regression or a constant luteal activity between pp d 21 and pp d 28, cows in either the GP or C groups that had CL on pp d 21 showed overall improvement of fertility. In contrast, fertility but not submission was reduced in GP cows failing to form CL prior to PGF treatment. In conclusion, the treatment with GnRH and PGF to commercially managed dairy cows as early as pp d 21 is able to improve the pre-required conditions for the early establishment of conception. However, management and/or other factors affecting conception and embryo-fetal development may play a limiting role to the improvement of fertility in hormonally treated dairy cows.
Table 6.1 Composition of the Total Mixed Ration
Component
High producer cow (kg/head)
Low producer cow (kg/head)
Corn silage 12 12
Grass silage 10 10
Alfalfa hay 1 1
Timothy grass hay 1 1.5
Beet pulp 1 0
Mineral mix 1 0
Koiwai dairy-21 14 12
Table 6.2 Changes in the endocrine status between 21 and 28 days postpartum in spontaneous ovulating cows and cows induced to ovulate with a GnRH and PGF2α protocol in a commercial dairy farm.
21 days postpartum 28 days postpartum Group Sub-group P4 (ng/ml) n (%) Sub-group P4 (ng/ml) n (%)
NCL 0.4 ± 0.1 19 (70.4) a NCL 0.3 ± 0.0 27 (54.4)
CL 4.7 ± 0.5 8 (29.6) x
C (n=47)
NCL 0.3 ± 0.0 7 (35)
CL 3.7 ± 0.4 20 (42.6)
CL 3.8 ± 0.7 13 (65)
NCL 0.3 ± 0.1 5 (17.2) b
NCL 0.3 ± 0.0 29 (60.4)
CL 5.2 ± 0.4 24 (82.8) y
GP (n=48)
NCL 0.4 ± 0.1 4 (21.1)
CL 3.1 ± 0.3 19 (39.6)
CL 2.9 ± 0.2 15 (78.9)
C: Untreated cows; GP: GnRH-PGF2α treated cows.
CL: Plasma P4 concentrations ≥ 1ng/ml; NCL: Plasma P4 concentrations < 1ng/ml.
a, b/ x, y
Number of cows within a column having different pairs of superscripts differed significantly (p<0.001).
Table 6.3 Metabolic parameters in spontaneous ovulating cows and cows induced to ovulate with a 7-day GnRH and PGF2α protocol started 21 days postpartum (pp) in a commercial dairyfarm.
GP: GnRH-PGF2α treated cows; C: untreated cows.
Metabolic GP group (n=48) C group (n=47)
parameter 21 days pp 28 days pp 21 days pp 28 days pp NEFA (mEq/L) 241.7 ± 17 232.2 ± 19.1 234.2 ± 17.0 227.4 ± 14.0 Glucose (mg/dl) 58.8 ± 1.0 60.0 ± 1.2 60.4 ± 1.0 61.3 ± 1.0 BHB (uM/L) 709.4 ± 64.1 740.8 ± 67.4 693.8 ± 37.0 697.1 ± 39.4 AST (IU/L) 84.2 ± 2.1 84.0 ± 2.0 85.8 ± 2.0 87.3 ± 2.4
Table 6.4 Fertility traits of cows induced to ovulate with a 7-day GnRH-PGF2α protocol started 21 days postpartum and spontaneously ovulating dairy cows in a commercial dairy farm. Culled cows were removed from analysis. Values are means (± SD) or percents.
Reproductive parameter GP group C group
Number of cows 48 47
Calving-to-first service (days) 91.2 ± 27.3 97.9 ± 33.9 First service conception rate (%) 21 (47.4) 24 (51.1) 100-d breeding submission rate (%) 35 (72.9) 29 (61.7) Calving-to-conception (days) 126.0 ± 51.9 131.4 ± 51.8 100-days conception rate (%) 17 (34.7) 16 (34.0) 150-days conception rate (%) 31 (64.6) 30 (63.8) Mean services/conception 2.1 ± 1.4 1.8 ± 1.3 GP: GnRH-PGF2α treated cows; C: untreated cows.
106
Table 6.5 Fertility traits of dairy cows differing in ovarian activity prior to and during the induction of ovulation with a 7-day GnRH-PGF2α protocol started 21 days postpartum in a commercial dairy farm. CL NCL CL NCL CL NCL C GP C GP C GP C 15 13 4 7 24 8 5 19 eter to-first service (days) 84 ± 20 100 ± 28 86 ± 30 86 ± 25 97 ± 32 113 ± 49 91 ± 20 94 ± 34 9 (60.0) 6 (46.2) 4 (100.0) 5 (71.4) 8 (33.3) 4 (50.0) 0 (0.0) 9 (47.4) breeding submission rate 14 (93.3) 6 (46.2) 3 (75) 6 (85.7) 14 (58.3) 4 (50) 4 (80) 13 (68.4) 120 ± 13bc 152 ± 14 ab 86 ± 25c 97 ± 20c 127 ±11abc 155 ± 17ab 174 ± 22a 118 ± 12bc 8 (53.3) 2 (15.4) 3 (75.0) 4 (57.1) 6 (25.0) 2 (25.0) 0 (0.0) 8 (42.1) 10 (66.7) 7 (53.9) 4 (100.0) 6 (85.7) 16 (66.7) 3 (37.5) 1 (20.0) 14 (73.7) nception 1.9 ± 0.3b 2.4 ± 0.4ab 1.0 ± 0.6b 1.3 ± 0.5b 2.1 ± 0.3b 2.0 ± 0.5b 3.6 ± 0.6 a 1.6 ± 0.3b GP: GnRH-PGF2α treated group; C: Control group. CL: Plasma P4 concentrations ≥ 1ng/ml; NCL: Plasma P4 concentrations < 1ng/ml. Means with different superscripts across columns significantly differ (p<0.05).
Chapter 7
Summary and conclusion
The present studies on the reproductive effects of the early induction of ovarian activity using GnRH and PGF2α in dairy cows investigated the ovarian, ovulatory and endocrine responses within the 7-day protocol for the synchronization ovulation using analogues of GnRH and PGF2α at two different stages in the early postpartum period. In addition, these studies assessed the influence of the type of response to the protocol started 21 days postpartum on the cyclic ovarian activity before the end of the voluntary waiting period under research and commercial farming conditions. Moreover, the influence of the metabolic status on the response to the treatment protocol started early in the postpartum (as on 21 days), and the effects of this early treatment combination on several fertility parameters of high producing dairy cows managed under commercial conditions were also studied.
1) Around the world, the dairy industry is afflicted by the continuous decline in the reproductive performance of milking cows despite several efforts that have been conducted to solve this problem. A considerable number of reports relate an early first ovulation with the improvement of the fertility. However, fewer reports have pointed the possibility of risking fertility by continuously exposing cows to progesterone early in the postpartum. While examining the way in which cows resume luteal activity postpartum, it was noticeable that in the majority of the cases a short luteal phase precedes the continuation of cyclicity.
The GnRH-PGF2α based synchronization of ovulation which is commonly practiced in cycling cows during a 7-day period is thought useful to mimic the resumption of ovarian activity. The hypothesis was that if started early in the postpartum,
the major effects of the treatments would be 1) the induction of the ovulation by the GnRH treatment, 2) the early regression of corpora lutea by the treatment with PGF2α
and the subsequent synchronization of ovulation of the new follicles that developed after the GnRH-induced ovulation and 3) the reduction of the possible adverse effects of a continuous exposure to P4 early in the postpartum. However, there was a lack of information on the specific ovarian and endocrine responses following the treatment with these hormones during the early postpartum period.
The present study found that the treatment with GnRH to dairy cows 21 days postpartum induced ovulation in a high proportion of cows. In addition, the ovulatory response was similar to that of cows treated later in the postpartum (37 days).
However, the ovarian morphology and the endocrine responses following a GnRH-induced ovulation were found different between cows treated early (21 days) than those treated later (37 days) in the postpartum period. Differences were also found between cows that have re-started cycling and those still anestrous. The larger number of recruited follicles found in anestrous cows after the treatment with GnRH on 21 days postpartum may have benefits for the in vitro embryo production such as ovum pick-up and IVF procedures. A GnRH-induced ovulation by the 3rd week postpartum may allow for the maximum utilization of genetic material before the re-start of breeding.
Despite the differences in ovarian morphology and in the hormonal response, the synchronized growth of new follicles and CL were also induced in a 7-day interval earlier in the postpartum. The competence of the induced follicles and CL was confirmed by the high degree of CL regression followed by ovulations after the treatment with PGF2α. The early re-establishment of cyclicity was then thought possible by treating cows with this protocol on day 21 postpartum.
2) The common selection of cows based on the milk production has been recently shown by several studies to delay the postpartum time to first ovulation. Based on the importance that an early resumption of the ovarian activity has on the subsequent fertility of cows, and also on the long time that would be required to solve this abnormality through genetic improvement only; the hormone treatment with a 7-day GnRH and PGF2α protocol started 21 days postpartum was carried out to investigate the response in the subsequent cyclic ovarian activity. The effects were studied under research and field conditions only after the treatment’s capacity to induce ovulation was confirmed (as presented in Chapter 3). The results of this study revealed that, while being investigated under experimental conditions, the treatment protocol effectively induced ovulation after GnRH treatment in non-cycling and cycling cows at comparable proportions (82% and 100%, respectively). Similarly, and based on the percentage of cows diagnosed non-cycling by 21 days postpartum that formed CL by 28 days (80%), ovulation following GnRH treatment was effectively induced under field conditions.
In comparison to the ovulation rate after the treatment with GnRH, the ovulation rate following PGF2α administration was reduced and occurred under both research and field conditions. In a small number of cows, an improper timing of the GnRH treatment (13% under research and 4% under field conditions) and a long period of ovarian inactivity (12% under field conditions) prevented a GnRH from inducing ovulation. As consequence, the presence of new dominant follicles and responsive CL by the time of PGF2α did not occur. Another reason for the reduction in the frequency of ovulations after the administration of PGF2α was the failure of CL to regress in response to the treatment. This event occurred only in some of the cows (33% under research and 69% under field conditions) in which the first postpartum ovulation was the result of the treatment with GnRH, and its frequency seemed to be increased in the field. Due to the
discrepancy of the luteolytic response observed between field and research conditions, the heterogeneity of the conditions under which cows were managed and also the possible differences in the pharmacokinetics of the different PGF2α analogues used are factors worth of consideration.
The enhanced ovulatory response after both GnRH and PGF2α treatments in cows that had ovulated by the time the treatment protocol started suggests that cows had CL in the early stages of formation. This may explain the maintenance of luteal activity during the 7-day treatment protocol. Therefore, this result reconfirms the importance of a sustained luteal activity to assure optimum synchronous results.
The treatment protocol reduced the time postpartum to the start of normal cyclic ovarian activity and increased the number of exposures to progesterone before the re-start of breeding in the cows that ovulated following PGF2α treatment. Similarly and based on the normal length of the estrous cycles, no evidence was found on the adverse effects that the failure of CL to regress after PGF2α treatment may have on the restoration of cyclic ovarian activity.
Moreover, it is reported herein for the first time that a 7-day treatment protocol using GnRH and PGF2α started 21 days postpartum is capable of inducing the beginning of a luteal phase around day 60 postpartum. This result could be a promising tool for non-estrus detection based breeding strategies such as time-AI protocols.
Presence of CL by 28 days postpartum and CL regression after PGF2α treatment are factors that have the potential to improve fertility in the early treated cows.
3) Based on the negative energy status reported to commonly occur during the early postpartum period in dairy cows, and also on the recent reports describing the direct effects that the different systemic metabolites related to the state of NEB may
have on the structures of the cow’s ovary; this study investigated the relation between the metabolic status within 3 weeks postpartum and the response to the induction of ovulation with GnRH and PGF2α. The studies were focused on cows differing in their cyclic status prior to the start of the treatments.
Most of the cows ovulated in response to the treatment with GnRH and the difference in cyclicity seemed to have no effect on this response. However, failure to ovulate following the PGF2α treatment was due to CL refractoriness and occurred in some cows first-induced to ovulate in the postpartum by the GnRH treatment. From soon postpartum to the time of GnRH treatment, these cows shared clear characteristics associated with factors reported to cause a long anestrous interval: higher concentrations of NEFA, lower Glu, and high BHB and AST levels at one and/or several observations during the first 3 weeks postpartum, as well as abnormally large sized follicles (27.8 mm). Based on the similar P4 concentrations by the time of PGF2α treatment between the cows with refractory CL and those in which CL did regress, this study considered that NEB may have adversely affected the period of follicular development and/or the subsequent process of luteal formation. The alterations caused by the NEB might have given origin to abnormal CL without affecting the steroidogenic capacity.
It can be concluded then that the treatment with a 7-day GnRH and PGF2α
protocol started 21 days postpartum can reduce the postpartum anestrous interval in normal cows and also in cows that according to a NEB status have the risk to re-start estrous activity late in the postpartum period. In addition, the metabolic status of dairy cows within 21 days postpartum is a limiting factor for the complete synchronous response to the treatment combination.
4) The cow has been the main focus of research on the several ways to
improve the reproductive performance. This tendency is most probably because is in the females where several factors may affect either the establishment of pregnancy and/or the viability of the conceptus during the period before parturition. The early re-establishment of normal ovarian cycles is of paramount importance because cows need to be bred early in order to calve at yearly intervals. However, failure to cycle and display estrus and suboptimal and irregular estrous cycles are some of the very important identified factors that depress fertility by lengthening the time to conception.
This study investigated the outcomes in the fertility following the treatment with a 7-day GnRH and PGF2α protocol started 21 days postpartum in dairy cows managed under commercial conditions. The high rate of cows without CL 21 days postpartum confirmed the recent tendency of delayed resumption of the ovarian activity in dairy cows. However, the enrolment of cows into the hormone treatment protocol significantly reduced the postpartum anovulatory interval by increasing the number of cows having a CL with normal steroidogenic function by 28 days postpartum. In comparison to the mean P4 levels observed 28 days postpartum for the cows in which GnRH induced the first postpartum ovulation in chapters 4 and 5, cows in similar conditions in this commercial dairy farm had improved P4 levels. This result suggested that the CL regression following the treatment with PGF2α and the subsequent ovulation and cyclicity of these cows was highly expected to occur. In fact and despite a possible limitation caused by initiating practices for breeding after 60 days postpartum in this farm, the overall 1) calving-to-first service interval (91 vs. 97 days), 2) the 100-day breeding submission rate (72.9 vs. 61.7%) and 3) the calving to conception interval (126 vs. 131 days) tended to be improved in the treated cows. However, according to our results the improvement in the parameters of fertility seemed to be regulated by the cyclicity status by 21 days postpartum. This is, cows having ovulated by this day would
have a more uniform improvement of fertility following treatment.
In conclusion, the treatment with GnRH and PGF2α as early as 21 days postpartum to commercially managed dairy cows is able to improve the pre-required conditions (e.g. early ovulation, early normal cyclic ovarian activity) for the early establishment of conception. However, management and / or other factors that affect conception and the posterior embryo-fetal development may play an important role when the improvement of the fertility is the target by hormonal treatments initiated early in the postpartum period.
The realization of this series of studies showed in detail some factors limiting the induction of ovulation, the differential ovarian response, and the ovarian cyclic activity and the fertility of dairy cows following the consecutive treatments with GnRH and PGF2α in a 7-day protocol started as early as 21 days postpartum. The enhanced follicular recruitment following a GnRH-induced ovulation by the 3rd week postpartum may be seen as an important finding to improve the utilization of genetic material from cows before the re-start of breeding. Moreover, the synchronized presence of both a follicular and a luteal phase near the end of the non-breeding period are important conditions for the development of new in-farm breeding strategies targeting the early establishment of conception to achieve one calf per year in dairy farming.
乳牛におけるGnRHと PGF2α製剤を用いた分娩後の 早期卵巣賦活化処置による繁殖成績向上に関する研究
カルロス サンテイアゴ アマヤ モント-ヤ
岐阜大学大学院連合獣医学研究科
（帯広畜産大学）
本研究は高泌乳牛において分娩後早期の GnRH-PGF2α処置による卵巣動態の 変化および内分泌学的反応について調べるとともに，その反応性と同時期の栄 養状態との関係について研究したものである。また，一般農家で飼育されてい る高泌乳牛にGnRH-PGF2α処置法を導入して，その効果を処置後の繁殖性をも とに評価・検討したものである。その結果，以下の成績を得た。
【１】分娩後21日および37日前後の乳牛（n=14）にGnRHを投与（d0）し，
その７日後(d7)に PGF2αを投与した場合の排卵，卵胞発育および黄体形成状 況と内分泌動態（P4，E2，FSH，IGF-1）を調べた。GnRH投与により全頭にお いて排卵が誘起された。また，GnRH投与時に黄体を有しない牛（GnRH21-NCL） では黄体を有する牛（GnRH21-CLおよび GnRH37-CL）に比べて，d0から d5 の血中P4濃度が低く，d2からd6のE2濃度が高く，d2-d4の卵巣内小卵胞数 およびd5の成熟卵胞数が多く，誘起黄体と新規卵胞は大きく，d0の血中 FSH 濃度は高かった。さらにGnRH投与から７日後のPGF2α投与によって黄体退行 とその後の排卵が誘起された。これらの結果から分娩後21日（あるいは37日 前後）のGnRH-PGF2α処置は効果的に分娩後の卵巣の賦活化を誘導できること が明らかになった（第３章）。
【２】分娩後21日の高泌乳牛にGnRH-PGF2α処置を施した（GP群，n=40） 場合の処置後の排卵性および卵巣の活性化状況を対照（C 群，n=55）のそれら と比較した。GnRH 投与時に黄体を有する牛（GP-CL と C-CL）と有しない牛
（GP-NCLとC-NCL）に区分したところ，GP-CL牛は供試牛の20〜27％，C-CL 牛は26〜34％に認められた。GnRH投与によってGP-CL牛では全頭，GP-NCL 牛では80〜82％の排卵率を得た。PGF2α投与後の排卵率はGP-CL牛では全頭，
GP-NCL牛では25〜55％であった。また，PGF2α処置により排卵がみられた牛 における処置後の正常発情周期への回帰は GP-CL 牛では分娩後 38〜39 日，
GP-NCL牛では37〜38日，C-CL 牛では40日であった。分娩後の第３回目黄 体期はGP-CL，GP-NCLおよびC-CL牛では分娩後60日前後にみられ，C-NCL 牛の77日に比べて有意に早かった。また，分娩後60日までの発情周期数にお いてもGP-CL，GP-NCLおよびC-CL牛では2.3〜2.8回と，C-NCLの1.5〜 1.9 回に比べて有意に多かった。さらに正常な発情周期の長さを有する牛の比 率は GnRH-PGF2α処置の GP 群では 82.0〜89.5％と対照の C 群の 47.0〜 61.5％に比べて高かった。以上の結果から，分娩後21日にGnRH投与，その7 日後にPGF2αを投与（GnRH-PGF2α処置）することによって，とくに分娩後長 期間に渡って排卵が認められない乳牛の早期排卵と卵巣賦活化を誘導できるこ とが示された（第４章）。
【３】分娩後早期のGnRH-PGF2α処置による卵巣の反応性と同時期の栄養状 態の指標となる代謝プロファイルとの関係について調べた。処置牛（GP 群，
n=15）と対照牛（C群，n=32）から分娩後１日，11日および21日に血液を採 取して血中Glu，NEFA，BHB，AST濃度を測定した。その結果，GP群において 処置前後の卵巣および内分泌動態と血中Glu，NEFA，BHBおよびAST濃度に相