ART : Assisted Reproductive Technology. The in vivo embryo production in cattle.

(1)

Dr. Ch. Hanzen, PhD

Retired professor of the Université de Liège

Consultant for the RUMEXPERT society

[email protected]

Publications :

http://orbi.ulg.ac.be/

Facebook :

https://www.facebook.com/Theriogenologie

Facebook :

https://www.facebook.com/RumeXperts/

Belgian Blue Group

: https://bbgforcrossing.com/

Training program on embryo transfer in cattle organized by BET Cipelang (Indonesia)

16 to 24th_{february 2018}

ART : Assisted Reproductive Technology

The in vivo embryo production in cattle.

(2)

1. General data in the World, Europe, USA, France and Wallonia

2. What’s an embryo : general data

3. How to induce the growth of several follicles ? The superovulation

4. Which factors can influence the results of a superovulatory treatment ?

5. How to collect the embryos ?

6. How to evaluate the quality of the embryos ?

7. How to freeze and to thaw the embryos ?

8. How to make the transfer of an embryo ?

9. What are the risk factors of an embryonic mortality ?

10. Which treatments to prevent embryonic mortality in the cow ?

Prof. Ch. Hanzen- La détection de l ’oestrus chez les ruminants

(3)

1. General data about the

embryo transfer

(4)

Importance of AI and ET according to the continent (Thibier 2005)

In Thibier M. / Thai J Vet Med. 2016. 46(4): 531-539.

1 cow out of five is concerned by AI

1 cow out of 500 is concerned by ET

(5)

(6)

Collections and embryos by collection in Europe (2016) Data from AETE

(2015 : 24 millions of dairy cows and 35 millions of beef cows (UE28)

(7)

Ovum pick up and number of embryo by OPU in Europe (2016) Data from

AETE (2015 : 24 millions of dairy cows and 35 millions of beef cows (UE28)

(8)

Number of transferred embryos obtained in vitro and in vivo in Europe

Data from AETE

(9)

Bovine embryo technologies in Europe

Data from AETE (2016) (http://www.aete.eu/)

(10)

Some data from France

19 millions of beef and dairy cattle (2016)

(11)

(12)

Some data (2016) from USA coming from 194 members of the

American Embro Transfer Association

(13)

(14)

(15)

Embryo transfer activity of AWE (2016) (4 people)

- % of flushings without any embryos : 8,1 %

- N of embryos by + flushings : dairy 4,7 and beef : 5,4

(16)

2. What’s an embryo ?

General data

(17)

LH induces the 1st meiotic division and the expulsion of the 1st polar body

Blockade at stage II of the meiosis

The fertilization induces the end of the 2nd_meiosis

and expulsion of the 2nd_{polar body}

1st polar body

2nd polar body

(Oogonie)

After some min

+ n

(18)

- http://www.embryology.ch/ (University of Lausanne)

- Senger Pathways to pregnancy and parturition 2005

2 cell

4 cell 8 cell morula

(19)

First steps of embryo development (Senger 2005)

(20)

(21)

Fig. 13-5

Hormonal regulation of the embryo : interferon tau (Senger 2005)

Interferon tau but also many others proteins secreted by the embryo and identified in the ULF (Uterine Lumen Fluid) Forde et al.

(22)

(23)

Progestero ne < 12 J R oxytocin Phospholipi ds Oestrogen s Arachidonic acid PGF2 a Phospholip ase Prostaglandine synthetase Ocytoci ne Contractio ns Luteal ischemia Apoptosis Synthesi s stimulati on

T

h

e

l

u

te

o

ly

si

s

(24)

Hormonal regulation of the embryo : inhibition of PGF2a synthetsis

due to the inhibition of oxytocine R (Senger 2005)

X

_X

No synthesis of PGF2a

(25)

Machaty et al. Production and manipulation of bovine embryos:

Techniques and terminology Theriogenology 78 (2012) 937–950

Ovoïd form : d12-13, Tubular form : d14-15, Filamentous form : d16-17

(Degrelle eyt al. Dev Biol 2005;288:448–60).

Quality of development depends of the quality of oocyte (Leroy et al. 2015)

Quality of development depends of the mother (Histotrophic effect) (Leroy et al. 2015)

(26)

Morphological changes of the embryonic vesicule

(Spencer et al. Reproduction 2004 DOI: 10.1530/rep.1.00398)

(27)

Elongation phase of the embryo

(Tveden-Nyborg et al. Reproduction 2005 DOI: 10.1530/rep.1.00850)

Ovine ovoïd embryo day 11 Ovine elongated embryo day 13

(28)

(29)

Organogenesis between day 25 and 40

Alberto et al. Development of bovine embryos derived from reproductive techniques.

Reproduction, Fertility and Development, 2013,25,907-917 H : heart; l :liver uc : umbilical cord e : eye t : tail fl : forelimb

(30)

50 days

54 days of pregnancy

http://loribovinesection.blogspot.be/2013/09/bovin e-fetal-anatomy-keywords-amnion.html

(31)

Evolution of the lenght of the

(32)

3. How to induce the growth of

several follicles ?

(33)

• High

genetic value

• Regularly cycled

(3 heats at 21 days intervals : the treatment can begin

between days 8 to 13 of the cycle)

• At least

two months after calving

• At least

40 days after a previous superovulation

treatment

• No vaccination or parasitic treatments during the two previous months

• Good

clinical examination

• No lesions of the vagina (pneumo or urovagina)

• No signs of uterine infections

• Normal size (<5 cm) of the cervix and uterine horns

• No ovarian abnormalities like cysts

• No adhesions at the uterine horns

• Body condition score > 2.5

(34)

• eCG

(PMSG) to stimulate the follicular growth. His use has been

abandonned due to his long half life (40 h) and persistence during 10

days : problems of anovulation overcomed by an injection at AI of

antibodies (not yet produced) to eCG and the decrease of the the

quality of embryos.

• Porcine pituitary extract of

FSH

to stimulate the follicular growth

• Very short half life : 5 hours

• Used with two daily injection during 4 to 7 days

• PGF2a

: to induce the luteolysis inducing an œstrus 36 to 48 h later

• GnRH

or

hCG

to induce ovulation (dominant or stimulated follicles

• Progesterone

to inhibit the development of a dominant follicle and an

œstrus during the ovarian stimulation

What hormones are used in a superovulation protocol ?

(Bo and Mapletoft Theriogenology 2014, 81,38-48)

(35)

2 9 8 7 6 5 4 3 17 10 16 15 14 12 12 11 18 20 19

One wave and one follicule

Dominant follicle LH dependent

FSH dependent

Recruited follicles Growing follicles

Gro wth pha se Static phase Atretic phase Atretic follicles Follicles < 8 mm Bollwein 2017 Dominant foll > 8 mm Bollwein 2017

(36)

1 2 3 4 5 6 7 8 9101112131415161718192021 0 0 -1 -2 -3 2 9 8 7 6 5 4 3 17 10 16 15 14 12 12 11 18 20 19 -3-2-101 2 3 4 5 6 7 8 9101112131415161718192021222324 2 9 8 7 6 5 4 3 17 10 16 15 14 12 12 11 18 20 19

Two waves cycle

Three waves cycle

Cycle lenght 21 days

Cycle lenght 24 days Wave Wave _Wave _Wave _Wave

(37)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 0 0 -1 -2 -3 mm Time of emergence of the 2nd_wave (Earlier if 3 waves)

Period of non dominant follicle (30 % of the cycle)

The window to begin a superovulation treatment is short

- Initiating gonadotropin treatments as little as 1 day before or after wave

emergence significantly reduced the superovulatory response (Adams et al. 1994) - The day of emergence of the second follicular wave has been shown to differ between two-wave cycles and three-wave cycles (Ginther JRF 1989)

2 9 8 7 6 5 4 3 17 10 16 15 14 12 12 11 18 20 19

(38)

One wave but several follicles

2 9 8 7 6 5 4 3 17 10 16 15 14 12 12 11 18 20 19

FSH injections or eCG injection (one)

Enough recruted follicles (> 2 mm)

Growth of several follicles if No negative influence of a dominant follicle Ovulation if no P4 PGF2a Puncture GnRH Oestradiol

(39)

Basic scheme of a superovulation protocol

0 11 13 14 8 FSH Reference heat PGF2a 15 16 AI AI 23 Flushing - TAI 48 and 72 h after PGF2a - 12 and 24 h after œstrus detection

And PGF2a after D 13 in cows

D 14 in heifers

Natural or induced by a pretreatment with IVD /PGF2a

Injected at the same time in recipient

9

Control of the presence of a dominant follicle and puncture if any

Control by US of the presence of enough (<15 vs > 25) small follicules (> 2 mm)

(40)

Stimufol

Name Stimufol Pluset Folltropin Folligon

Society Reprobiol Calier Vetoquinol

Total pFSH 500 mcg 500 UI 400 mg (700 UI) eCG (5 mcg/ml) (50 UI/ml) (20 mg/ml) Total pLH 100 mcg 500 UI < 1000 µg (1 mcg/ml) (50 UI /ml) Solvant (ml) 10 20 20 D1 8h 1,8 3,0 2,5 2-3000 20h 1,8 3,0 2,5 D2 8h 1,5 2,5 2,5 20h 1,5 2,5 2,5 D3 8h 1,0 1,5 2,5 20h 1,0 1,5 2,5 PGF2a : cows D4 8h 0,6 1,0 2,5 PGF2a : heifers 20h 0,6 1,0 2,5 D5 8h AI / anti-eCG 20 h AI D12-D13 Recovery/PGF2a Total (ml) 10,0 16,0 20,0

Superovulation protocol

with œstrus detection

(41)

Donor Recipient 8h 8h 8h 20h 20h 8h 8h D1 P4 + PGF2a D5 P4 + GnRH D8 GnRH D9 FSH D10 FSH FSH D11 FSH FSH D12 FSH FSH PGF2a P4 - PGF2a D13 P4 - FSH PGF2a D14 : oestrus GnRH FTAI GnRH or hCG D15 FTAI

D21 Flushing PGF2a Transfer

Superovulation protocol and synchronisation of the

recipient without œstrus detection (Bos taurus)

(Baruselli et al. Theriogenology 2011, 76,1583-1593)

• PGF2a D1 to induce the luteolysis of the possible CL • D8 GnRH to induce a follicular wave

(42)

What kind of hormonal protocol can we recommand to induce a

superovulation in Bos taurus (beef and dairy) and Bos indicus ?

Fo rb id d e n in E u ro p e Intermediate doses of FSH Low doses of FSH High doses of FSH

(43)

GnRH

can replace the injection of oestradiol in Bos taurus ?

(44)

1. Problems links to the use of porcine FSH/LH

• a potentially high and variable FSH⁄luteinizing hormone (LH) ratio • The optimal ratio need to be identified (15 to 20 % of LH ? )

• batch-to-batch inconsistencies

• 60 isoforms of FSH and LH have been identified within the pituitary • potential disease transmission

• _An_{increased amount of LH}_{early in the superovulatory protocol may}

activate the LH receptor (LHR) leading to premature luteinization, resulting in early ovulation of the follicles and ultimately leading to decreased

fertilization rates and embryo production.

• _{LH present within the FSH preparation may}_interfere_{with the effectiveness} of FSH.

What kind of FSH ?

(45)

How to avoid the necessity to detect œstrus or to select the best

moment of the cycle to induce a superovulation ?

1. By injecting 2.5–5 mg estradiol-17b (forbidden in Europe) at the time of insertion of an intravaginal P4 device

• The oestradiol suppresses FSH and after metabolization of oestradiol a new surge of FSH and a new wave of 3-5 mm follicules appears (4 days later on average : time to begin the FSH treatment)

• The P4 contributes to the growth of a more uniform group of viable follicles with competent oocytes

2. By eliminating the suppressive effect of the (two biggest follicles : to be sure) dominant follicles using ultrasound-guided follicle to puncture the two

largest follicles to ensure that the dominant follicle was removed.

Superstimulatory treatments are then initiated 1 to 2 days later, at the time of emergence of a new follicular wave.

3. By injecting a GnRH to induce the ovulation of the dominant follicle and the appearance of a new wave 1 to 2 days later. But ovulation appears only in 44% to 54% of dairy cows, 56% of beef heifers and 60% of beef cows.

(46)

4. By beginning the superovulation treatments at the time of the emergence

of the 1st_{wave but we observe a reduction of the number of transferable}

embryos

5. By injecting small doses of FSH (Twice daily for two days) or 400 /500 IU eCG 2 days before the beginning of superovulation treatment : such injections could induces the growth (1-2 mm/d) of small (1 mm) follicles to reach 3 or 4 mm.

6. By lengthening the FSH treatment protocol to 7 days, without increasing the total amount of FSH administered. Such treatment can to recruit more small follicles into the follicular cohort available for superstimulation, and provide the additional time needed for these follicles to reach an ovulatory size and acquire the capacity to ovulate (2,5 times more transferable embryos have been obtained : Dias et al. Theriogenology 2013,79,1177)

(47)

Garcia-Guerra et al. Theriogenology 78 (2012) 353

The long treatment with FSH (400 mg in 14 injections vs 400 mg in 8 injections) don’t increase the number of follicles present at LH injection (33.4 ± 4.4 vs 26.7 ± 3.2), neither the ovulating diameter (11.5 ± 0.2 vs 11.7 ± 0.3) but

increase significantly (P<0.0001) the % of ovulation of these follicles (93 ± 1.8 vs 66 ± 6.9) that ovulated between 12 and 36 h after pLH injection (%)

No significant effect on

(48)

Growth of multiple follicles

Ovaries after superovulation

(Corpus luteum)

(49)

• 0–60 recovered embryos per donor (Hasler 2003)

• 6,3 transferables embryos

by flushing (Perry 2013)

• 20% of donors producing no transferable embryos

(Hasler 2003)

• 90 % of ovulations appear during 22 hours (Yadav et al. Therio

1986,26,509).

• The interval between the first and last ovulation is between

1,2 et 12

hours

(Adams Therio 1994,41,19; Purwantara et al. nim.Reprod.Sci.

1994,37,1; Laurincik et al. Therio 1993,39,537)

• Interval between luteolysis and œstrus (2xPGF2a) : 39,4 ± 7,7 h (23,4

to 58,3 h).

• The

duration of œstrus

is 13,2 ± 4,1 heure (5,4 à 20,9 h) (Dalton et

al. J.Anim.Sci.,2000,78,2081)

(50)

4. Which factors can influence the

results of a superovulatory

(51)

Name Pluset Pluset Folltropin Folltropin

Cows (34 %) Heifers (66 %) Cows (20 %) Heifers (80 %)

Total UI pFSH 850 500-600 630 420-490 Ayrshire _{78 %} 22 % 1398 59 % 41 % 2592 Holstein N superovulations

Eight injections (declining doses) beginning between d 9 to 12 after a standing estrus (4 days) PGF2 was injected at sixth (cows) or seventh (heifers) FSH treatment

Double AI 9 to 15 h apart (13 % with sexed sperm)

at 48 h for heifers and 60 h for cows (after PGF) for the 1st AI

N embryos 7.172 12.228

% Fresh / % Frozen 20/80 35/65

% Grade 1,2 and 3

fresh embryos 46 %, 38 %, 16 % 53 %, 33 %, 14 % Transfer of fresh

embryos Heifers 56 % and cows 44 % Heifers 79 % and cows 21 %

Influence of the FSH

Folltropin (FSH + LH)

Vs Pluset (FSH + LH)

(52)

• No significative difference between Folltropin and Pluset of the % of

unsuccessful embryo collections (no transferable embryos)

• Heifers : 11.5% (Group F) and 14.8% (Group P)

• Cows : 15.6% (Group F) and 15.7% (Group P)

• No significative difference in the % of low responder (< 5 CL)

• Heifers : 6.4% (Group F) and 5.2% (Group P)

• Cows : 11.3% (Group F) and 8.3% (Group P)

• No significative difference in the % of quality grade (1 excellent or

good and 2 : fair) and developmental stage (4 to 9) (5 early

blastocyst and 6 : blastocyst)

Influence of the FSH

: Some results : Mikkola et Tapponen 2017

(53)

S

S S

Folltropin vs Pluset in heifers and cows (number of structures)

Influence of the FSH

Folltropin (FSH + LH)

Vs Pluset (FSH + LH)

S

(54)

Is it possible to

reduce the number of FSH injections

?

(In Bo and Mapletoft Theriogenology 2014, 81,38-48)

Objectives

•To reduce the number of manipulations •To reduce the stress of injections

•But the the half-life of pituitary FSH is short in the cow (5h) (Laster et al. 1972) What kind of possibilities

•A single subcutaneous administration of FSH induces a superovulatory response equivalent to the traditional twice-daily treatment protocols in beef cattle in high body condition but results were not repeatable in Holstein cows, which had less subcutaneous adipose tissue (Bo et al. 1994)

•The response is improved in Holstein cows when the single injection was split into two : 75% of the FSH dose was administered subcutaneously on the first day of treatment and the remaining 25% was administered 48 hours later, when

PGF2a is normally administered (Lovie et al. 1994)

•Good results can be obtained with two SC injections (48 h apart) of FSH diluted in 10 ml of an 0.5 or 1 % hyaluronane solution (Tribulo et al. 2012, Hasler et al. 2012)

(55)

Effects of lactation and season on the embryo production in large dairy herds (Chebel et al. Theriogenology 2008, 69,98-106)

702 superovulations of dairy cows (12.272 kg/305 d)

8 injections of FSH in 4 days, PGF and GnRH 48h after PGF2a, AI 12 and 24h after GnRH

P<0.005

10.9 ± 0.4 ova/embryos

Correlation with interval between superovulation

(once = 11.0 ± 0.7, <60 days = 8.6 ± 1.2, > 60 days = 12.4 ± 0.9; P = 0.03 No effect of brand of FSH

4.7 ± 0.2 viable embryos

(56)

Effect of THI and number of lactation on the embryo production after superovulation in large dairy herds (Ferraz et al Theriogenology 2016, 86,1834-1846)

516 superovulations of dairy cows (11.000 kg/305 d) (< 120 d PP)

8 injections of FSH in 4 days, 2 PGF 12h apart , AI 12 and 24h after œstrus detection

No effect of calving problems

(dystocia, fetal death, twin gestations, PR) or puerperal metritis

(57)

Factor influencing the response to superovulation :

the ovarian follicular population

1. Cow can be divided in two groups (Monniaux et al. 1983) : The first group has 50 to 200 follicles (> 0,6 mm) (> 600 if good responders) and the second group has a huge number of follicles who undergoes atresia at the time of superovulation treatment .

2. The number of follicles into a wave is very variable between cows : 8 to 56 after recruitment (Burns et al. 2005)

3. The stimulatory response is different in cows with > 30 follicles and in cows with < 30 follicles at the treatment (Singh et al. 2004) or between beef cows having < 15 follicles (> 2mm) and cows with > 25 follicles at the wave

emergence (Ireland et al. 2007). 4. Practical interest

• _{Ultrasonography}_{could be done at the beginning of FSH treatment}

• _{Evaluation of}_{Anti Mullerian hormone}_{(glycoprotein produced by small} growing follicles (Monniaux et al. 2013, Rico et al. 2012)

• _{Increase the}_{number of FSH}_{(during 6 to 7 days) or inject}_eCG_{at the end} of treatment

(58)

Why to inject

eCG

at the end of FSH injections

• FSH

is required for follicle recruitment and growth (Adams et al. 1992),

until the dominant follicle reaches 8.5 mm in diameter in Bos taurus

breeds of cattle (Ginther et al. 1996) and 6.2 mm in Bos indicus

(Sartorelli et al. 2005).

• After selection the growth of dominant follicle becomes LH dependent

(Mihm et al. 2008).

• eCG is a gonadotropin with FSH and LH activity (Murphy and Martinuk

1991).

• The treatment in Bos indicus cows with two injection of 200 IU of eCG

after three days of treatment with FSH increases the number of

ova/embryos and increased the number of transferable embryos

mainly when the produced average to below average numbers (Barros

et al. 2008; Mattos et al. 2011)

(59)

At which

frequency

can be repeated a superstimulation ?

What’s the delay of luteal regression after the injection of PGF2a

•At flushing : 3.6 ± 0.8 days (88% of estrus within 7 days)

(Mapletoft et al. 1991).

•At flushing and 1 day later : 4.1 ± 0.6 days vs. 5.3 ± 2.2 days (no

differences in the interval to estrus or to ovulation (Garzon et al. 2013)

The interval to ovulation depends on the number of CL (Cabra et al. 2011)

•4.0 ± 0.5 days in cows with 1 to 2 CL,

•7.6 ± 1.5 days in cows with 3 to 4 CL

•10.4 ± 0.9 days in cows with 5 to 10 CL

•11.7 ± 0.6 days in cows with 11 to 15 CL

•10.8 ± 0.8 days in cows >15 CL

(60)

Has the

interval between superovulations

an influence on the

quality of embryos?

No significative difference

(61)

5. How to collect the embryos ?

The collect of embryos in the Charolais cow

(Dr Escouflair)

(62)

Step 2 : epidural

Step 3 : Introduction of the catheter Step 2 : cleanliness of the vulva

Step 1 : restraint of the animal

Cervix Dilator, stainless steel www.minitube.com

(63)

Left horn Right horn Inflated balloon

(Air/liquid) In the last inferior

third of the horn

The medium goes out and goes in (with the embryos) by the same way

Recovery of the embryos

with a two way catheter

Cervix Left horn Right horn Embryo-Flushing-Catheter-Two ways www.minitube.com) Recovery of medium with embryos Injection of medium

(64)

Prof. Ch. Hanzen- La détection de l ’oestrus chez les ruminants Left horn Right horn Cervix Inflated balloon (Air/liquid) In the last inferior

third of the horn

Injection of medium

Recovery of medium with embryos The medium ( and the embryos) goes out at

the end of the horn and is got back on the base of the balloon

Recovery of the embryos

with a three way catheter

Embryo-Flushing-Catheter

(6 or 8 mm diameter) Three ways (www.imvtechnologies)

(65)

(66)

Step 4 : inflation of the balloon

(67)

Some medium to flush the uterine horns

Medium to collect and preserve the embryos (IMV technologies : https://www.imv-technologies.com/ ) EmXcell or Euroflush (with Ab and BSA)

EMCARE™ Holding Solution

http://icpbiorepro.com/emcare-embryo-transfer-products.html Vigro embryo transfer media

http://www.eggtech.co.uk/resources/

300 to 400 ml by uterine horn If two ways catheter

3 times 20 ml 3 times 30 ml 3 times 40 ml

In case of cows the volume can be increased

(68)

Step 6 : the recovered medium

(with the embryos) is poured in the filter

Step 7 : the filter is flushed with the medium

NB : the embryos can be also identified into the 50 ml obtained by decanting (10 min) the flushing medium. Such method is less expensive and the medium is more free of cells debris or mucus

Filter for embryos www.minitube.com

(69)

Step 8 : the embryos are researched

with a binocular loup (magnification x 45-60)

Micropipettor for handling of embryo Step 9 : the embryos are selected according to their quality and displaced with a micropipettor into small dishes for washing

Step 10 : after washing, the embryos are placed during 10 to 12 min into the ethylene glycol before freezing

(70)

Some companies who provide material

https://www.imv-technologies.com/

http://www.eggtech.co.uk/

https://www.minitube.com/

(71)

6. How to evaluate the quality

of the embryos ?

(72)

• Morphological criteria

• External diameter of the blastocyst : between 14 and 200 microns

• Sphericity, thickness, cracked aspect of the membrane pellucida

• Ease of identification of trophoblast, inner cell mass and blastocoele

• Number of cells with same size and regularity of the wall

• Presence of cells in the perivitellin space

• Hatching modality

• Metabolic criteraia

• Enzymatic test : the Fluorescéine Diacétate is transformed by

esterases in an non fluorescent compoiund)

• Evaluation of the glucose consumption

• Evaluation of the syntheis of lactate deshydrogénase

(73)

(74)

(75)

• Code 1: Excellent or Good (freezable embryos, international trade). • Symmetrical and spherical aspect

• Individual blastomeres are uniform in size, color, and density

• 85% of the cellular material should be an intact (no or few extruded material in the perivitelline space.

• The zona pellucida should be smooth and have no concave or flat surfaces of the zona pellucida (that might cause the embryo to adhere to a petri dish or a straw). • Code 2: Fair Transferable but not freezable embryos.

• Moderate irregularities in the overall shape of the embryonic mass or in size, color, and density of individual cells.

• Delay of development according to the stage of transfer • At least 50% of the embryonic mass should be intact. • Code 3: Poor (not transferable or freezable)

• Major irregularities in shape of the embryonic mass or in size, color, and density of individual cells (wall of the cell are difficult identify).

• 25% of embryo mass must be intact. • Code 4: Dead or degenerating.

Evaluation of the quality of the embryo : 4 grades

(76)

Day 0

1

2

3

4

5

6

7 ₈

₉

Chronological development of the embryo In Winterberger -Torrres et Sevellec 1987

(77)

(78)

Gfmer.ch

N0 1 Unfertilized oocyte

(79)

N0 2 Stages 2 – 12 cells

2 cells 4 cells

(80)

N0 2 Stages 2 – 12 cells (Quality 4)

Philips et Jahnke 2016 Philips et Jahnke 2016

(81)

N0 3 Early morula (40 Blastomeres)

(Bo and Mapletoft Anim Reprod 2013)

- A mass of at least 16 cells.

- Individual blastomeres are difficult to discern from one another.

- The cellular mass of the embryo occupies most of the perivitelline space. 16 cells

(82)

N0 4 Compact morula

(Bo and Mapletoft Anim Reprod 2013) - I n d iv id u al b la st o m e re s h av e c o ale sc e d , fo rm in g a c o m p ac t m as s. - T h e e m b ry o m as s o cc u p ie s 6 0 to 7 0 % o f th e p er iv ite llin e s p ac e.

(83)

N0 4 Compact morula quality 2

(84)

A morula in a needle’s eyes (for comparison)

(85)

N0 5 Early blastocyst

- An embryo that has formed a fluid-filled cavity or blastocele and gives a general appearance of a signet ring.

- The embryo occupies 70 to 80% of the perivitelline space.

(Early in this stage the embryo may appear of questionable quality because it is difficult to differentiate inner cell mass from

(86)

(87)

N0 6 Blastocyst Quality 1

- _{Pronounced differentiation of the outer trophoblast layer and of the} darker, more compact inner cell mass is evident.

- _{- The blastocele is highly prominent,with the embryo occupying most of} the perivitelline space. Visual differentiation between the trophoblast and the inner cell mass is possible at this stage of development.

(88)

N0 7 Expanded blastocyst

- _{The overall diameter of the embryo dramatically increases}

- _{There is a concurrent thinning of the zona pellucida to approximately} one-third of its original thickness.

(89)

(90)

Evolution of the diameter of the embryo

(91)

N0 8 Hatching or hatched blastocyst

- Embryos can be undergoing the process of hatching or may have completely shed the zona pellucida.

- Hatched blastocysts may be spherical with a well defined blastocele or may be collapsed. Identification of hatched blastocysts can be difficult

(92)

(93)

6. Free blastocyst

What is

it ?

Blastoc

yst

(101)

Where is (are ) the worse ?

https://www.evolution-xy.fr/fr/actualite/catalogue-vente-embryons-races-holstein-normande

(102)

7. How to freeze and to thaw

the embryos ?

To vizualize the procedure (English)

(103)

• STEP 1 : Segregate (X10) the specimens from one donor cow into separate groups (unfertilized ova, degenerate embryos, or transferable quality

embryos) : only embryos grade 1 and 2 can be freezed

• STEP 2 : Inspect (x50) quality grade 1 and 2 to ensure that the zona pellucida of the embryo is intact and that there is no material (e.g., cells, mucus)

adhering to the zona pellucida (Any embryo with a cracked or missing zona pellucida or with a zona pellucida having adherent material should be

discarded ).

• STEP 3a : washing of the embryos into 10 washing solutions (not more than 10 embryos at one time) each solution is diluted at 1:100 of the previous

solution).

• STEP3 b : If transmission of viral diseases is of concern, wash embryos two times in a 0.25% trypsin solution (< 90 sec for the two times) then 5 times more in embryo isotonic washing medium (diluted at 1:100 of the previous solution).

(104)

• STEP 4 : Equilibration step (penetration of the cryoprotective agent (CPA) into the embryo) by transfer of the embryo from the isotonic embryo holding

medium into a hypertonic solution (1.4-1.5 Molar concentration) of a CPA : (Equilibration time with ethylene glycol : 5 min and with glycerol : 10 min).

• STEP 5 : loading the embryo into a straw (can be done during the step 4) : with or without sucrose with bubble air (see the figure)

• STEP 6 : Placing the embryos for at least 2 min into a controlled freezing machine whose temperature has been cooled from ambient temperature to -6°C/-6.5°C.

Steps to freeze the embryos (Youngs CR JOVE 2011)

(105)

(106)

In 2009 (AETA) more than 99% of beef and 94% of dairy

embryos in the United States were frozen in ethylene

glycol.

The downside of the glycerol is that upon

thawing the embryos had to be removed from straws and

rehydrated through a series of sucrose dilutions and then

reloaded into straws for transfer to recipients.

(107)

Sucr ET ET ET Sucr CP

CP A A A A Cap

13 mm

Straw to freeze an embryo (with or without sucrose)

Sucr : Sucrose (0,25 M) A : Air

ET : Ethylene glycol CP : cotton plug end

OCM : Ovum culture medium : Embryo OCM ET ET CP CP _A Cap Identification OCM ET A A A

NB : the use of long cap increase the risk to broke the straw when she is taken from the container

(108)

• Rapid freezing or Vitrification : high cryoprotectant (DMSO, polyethylene glycol, glycerol) concentrations (25 %) and very rapid temperature

decrease to 196°C to freeze the embryos avoiding the ice crystal formation. • Slow freezing process using lower molar concentration of cryoprotectant

(glycerol or ethylene glycol) with or without the use of sucrose (not going into the cell at the difference with glycerol and ethylene glycol) designed to slowly replace the intracellular water with cryoprotectant and by

increasing the osmotic pressure extracellularly, reduces the ice crystal formation.

• Remark : The cryoprotectant agent decrease the freezing point (see the seeding to increase this freezing point)

• Some are going inside the cell : glycerol or ethyleneglycol, the others not : sucrose who has an osmotic effect (water goes out the cell)

(109)

• STEP 7 : seeding (to induce ice crystal formation in the CPA solution inside the straw ) by using a pair of tongs supercooled in liquid nitrogen to touch the

column of CPA solution either above or below the embryo.

The water in the CPA solution will crystallize in the region exposed to liquid nitrogen, and ice crystals will spread to the column of CPA solution

immediately surrounding the embryo (see the opalescent colour).

Hold embryos at seeding temperature for 10 minutes before further cooling.

Steps to freeze the embryos (Youngs CR JOVE 2011)

Opalescent colour

(110)

Seeding

Portable incubators www.minitube.com

(111)

• STEP 8 : continuing the dehydratation of the embryo by cooling the embryos at a rate of 0.5°C/min down to a temperature of -34°C.

• Hold embryos at -34°C for 10 minutes before plunging embryos into liquid nitrogen (-196°C).

• Place cryopreserved embryos into an appropriately labeled goblet (filled with liquid nitrogen) attached to an appropriately labeled cane

• Place the cane into a canister of a liquid nitrogen dewar for short- or long-term storage.

Steps to freeze the embryos (Youngs CR JOVE 2011)

(112)

Step 4 equilibration and loading the embryo

Step 7 Seeding : induction of crystalization

Step 8a : Continuing the dehydratation until – 34°C

Step 8b : Hold embryos at-34°C for 10 min and plug them into nitrogen liquid

Stockage Very quick thawing (2000°C/min)

(113)

• STEP 9 : thawing

• Pull the canister into the neck of the liquid nitrogen dewar, ensuring that the canister remains below the frost line in the dewar.

• Remove the straw from the goblet avoiding rewarming the others straws

• Hold the straw in air for 3-5 seconds (to reduce the incidence of a cracked zona pellucida)

• Submerge the straw into a 37°C water bath for an additional 25-30 seconds.

• Wipe the straw avoiding to erase the embryo identification on the straw

• Cut the non-cotton plug end of the straw (if sealed using heat or PVC powder)

or carefully remove the plastic sealing cap

(114)

• STEP 10a : Loading the straw into an embryo transfer device and transfer as quickly as possible to a synchronous embryo recipient (but ONLY if the

embryo was cryopreserved using ethylene glycol) = quick thawing • STEP 10b : = slow thawing

• Hold the open end of the straw over a dish containing a 1.0 Molar concentration of sucrose (a non-permeating compound),

• use a pair of scissors to cut off the cotton plug end of the straw.

Contents of the straw should freely flow into the sucrose solution, but pushing a small column of air through the straw may be necessary if any residual medium exists inside the straw.

• Allow embryos to remain in sucrose solution for 10 minutes, and then transfer embryos into isotonic embryo holding medium for 10 minutes. • Evaluate embryos post-thaw, load into a new straw, and transfer to

suitable recipient females.

(115)

Last step :

(116)

8. How to make the transfer

of an embryo ?

A definition

Transfer of an embryo, derived from the mating of genetically proven, valuable parents, into a fertile but less valuable

recipient (host) female who carries the pregnancy to term & offspring to weaning

(117)

Species Year

Reference

---Mouse 1971

Whittingham 1971

Cow

1973

Wilmut et Rowson 1973

Rabbit 1974

Bank et Amurer 1974

Ewe

1974

Willadsen et al. 1974

Rate

1975

Whittingham 1975

Goat

1976

Bilton et Moore 1976

Horse 1982

Yamamoto et al. 1982

Women1983

Trounson et Mohr 1983

Sow

1991

Kashiwazaki et al. 1991

(118)

• Locoregional anesthesia (2 ml procaïne by epidural, max 4 ml)

• Day 7 after estrus : High level of synchrony between donor and recipient (1 day)

• Very cleanliness of the manipulations (progesterone dominated uterus is susceptible to infection)

• Transfer to uterine horn on the same side as the corpus luteum • Into the ispsilateral (related to the CL) horn as deep as possible

• Highly skilled to gently manipulate the cervix (avoid the release of PGF2a) and to avoid trauma to uterine endometrium (blood). In < 60 sec ( (PR < 60 sec : 57 % vs 21 % if > 60 sec according to Jaskowski et Urbaniak 2006). On average the time to pass the cervix is 95.1±62s (Czeladko et al. 2009

http://www.aete.eu/index.php/publications-aete/proceedings/82-aete-proceedings-2009/file

• Thawing of the straw during 6 to 10 sec at air and after during 30 sec in a water bath at 22 to 25°C (if freezing with ethylene glycol)

• Rewarming of the gun •

(119)

• During dioestrus the mature CL reaches a maximum size of 20 to 30 cm

between days 6 and 18 after ovulation. The consistency has been described as "liver-like" .

• An ovulation papilla or crown protrudes from the surface of the ovary. At the stage of maximum development, the CL often occupies more space than the remainder of the ovarian tissue and has a clean outline.

• The CL of pregnancy is rounded and embedded in the ovary. Sometimes, it feels more rounded and smooth than the cyclical CL.

• A persistent CL is never observed except in pregnancy or where there are uterine lesions such as pyometra or congenital segmental hypoplasia (e.g. uterus unicornis).

• Corpora lutea with a fluid filled central cavity (lacuna) have been observed in an average 30 %

(120)

The

detection of the CL (and its size)

: a key point of embryo transfer

(Yoshida et al. Animal Science Journal 2012,83,207-212)

Corpus luteum Diam CL (mm) P4 (ng/ml) (n=543)CL (%) (n=370)PR I 22,9 ± 3,0 8,9 ± 2,2 32,2 76,0 II 19,7 ± 2,4 6,9 ± 1,8 42,0 74,6 I + II 20,1 ± 3,1 8,1 ± 2,3 74,2 75,2 III 15,4 ± 1,1 4,6 ± 3,0 24,3 47,7 IV 6,4 ± 5,6 3,5 ± 2,5 1,5 50,0 III + IV 12,4 ± 5,4 4,0 ± 2,8 25,8 47,9 Total 100 68,1 CL I : >= 20 mm CL II : >= 20 mm with a cavity CL III : >=15 mm and < 20 mm with or without a cavity

CL IV : < 15 mm

(121)

(122)

(123)

Size of the corpus luteum and P4

(Luttgenau and Bollwein Reproductive biology 2014,14,103-109)

(124)

Magnetic Resonance Image

Attributes of the Bovine Corpus

Luteum During Development and

Regression

(Hilton et al. The Anatomical

Record 2003, 272A:454–459)

(125)

The color Doppler offers some new perspective to evaluate the quality

of the follicule and the subsequent corpus luteum (de Tarso et al. Reproduction, Fertility and Development, 2017, 29, 448–457)

 The dimensions of preovulatory follicle (POF) and the CL are highly correlated with the blood flow of both structures and with P4 concentrations produced by the resultant CL.  The evaluation of POF blood flow could be an appropriate approach to estimate the

(126)

Factors affecting success of embryo transfer in large dairy herds (Chebel et al. Theriogenology 2008, 69,98-106)

702 superovulations of dairy cows (12.272 kg/305 d)

8 injections of FSH in 4 days, PGF and GnRH 48h after PGF2a, AI 12 and 24h after GnRH

1183 frozen embryos transferred on heifers (œstrus detection) or cows CIDR/Ovsynch protocol)

(127)

• Embryobec (Québec)

• Quality 1 (n = 3252) : 61,7 % • Quality 2 (n = 4649) : 59,0 % • Quality 3 (n = 2090) : 50,3 % • Mean (n = 991) : 58,3 %

The quality of embryo

influence the results

Rodrigues et al. Reprod Dom Anim. 2018;53:152–156.

(128)

Factors affecting success of embryo transfer in large dairy herds (Ferraz et al Theriogenology 2016, 86,1834-1846)

516 superovulations of dairy cows (11.000 kg/305 d) (< 120 d PP) 8 injections of FSH in 4 days, 2 PGF 12h apart ,

AI 12 and 24h after œstrus detection 10.297 embryos transferred

in cows (> 120 d PP) and heifers 6 to 9 d after oestrus detection

P<0.05 for different subscript Negative effect of puerperal metritis 35,7 vs 30,2 % (P<0.05)

(129)

Importance of the

synchronization

between the uterus of the donor and

the uterus of the recipient (Janowitz : Animal Breeding Abs 1994 : 2478)

% of PR

The pregnancy rate is higher when the recipient is in œstrus

1 day before or at the same day that the donor cow

-Rodrigues et al. Reprod Dom Anim. 2018;53:152–156.

-Kenyon et al., Animal Reproduction Science, 2013, 136, 223–230

(130)

Some influencing factors (Embryobec Quebec, Canada)

http://www.embryobec.com/

NL

Heifers

Cows

56,4 (9.358) 49,9 (4.332)

Signs of

estrus

Mucus

observed

No mucus

observed

56,2 %

(10.508)

50,6 %

(1.057)

Type of heat Natural

Induced

50,8 %

(3.868)

54,1 %

(16.172)

Stage of

embryo

Morula

Early blasto

Blasto

(131)

Non return rate Calving rate

Heifers Cows Total Grade 1 Grade 2 Grade 3 Total Folltropin 63,2 65,9 64,2 60,3 58,9 52,3 41,8 Pluset 60,1 65,5 62,4 63,6 57,0 52,3 40,4

Effect of

FSH and age

on the pregnancy and calving rate

(19.400 embryos, 16 years)

No significative differences

(132)

9. What are the risk factors of

an embryonic mortality ?

(133)

Before Fecundation D 1 to _D7 D8 to D27 D28 to D60 D61 to _D90 Non Fecund 15 à 30 % Very early EM 30 à 50 % Early EM précoce 20 % Late EM 12 % Early abortion < 2 % Embryo Foetus

Embryonic mortality (EM) during the first trimester of

pregnancy (Wiltbank et al. 2016)

(134)

Before Fecundation D 1 to _D7 D8 to D27 D28 to D60 D61 to _D90 Non Fecund 15 à 30 % Very early EM 30 à 50 % Early EM précoce 20 % Late EM 12 % Early abortion < 2 % Embryo Foetus

Embryonic mortality (EM) during the first trimester of

pregnancy (Wiltbank et al. 2016)

(135)

Reynolds Jim, DFCC, Veterinary Medicine Teaching and Research Center, (2016) 22,2 23,3 24,4 25,6 26,7 27,8 28,9 30,0 31,1 32,2 33,3 34,4 35,6 36,7 37,8 38,9 40,0 41,1 42,2 43,3

Sugiono et al. 2016 Indonesia (Blitar district)

(136)

Heat stress has a detrimental effect on the preimplantation embryos

Heat exposure of 40.0°C to 42.0°C significantly decreased

developmental competence of 1-to 8-cell stage embryos, but showed little to no effect at morula

and blastocyst stages

Oxidative stress originating from the elevation of temperature could be the main factor inhibiting normal embryonic development

Prevention by antioxydant would result in the survival of embryos under heat stress.

1

2

(137)

Significative effect of season and breeding technique

P<0.001 on CR and PL

Compared to AI (n=19.112) , the embryo transfer (n = 5.364) increases the PR during hot seasons (Holstein dairy cows in Brazil) and in repeat-breeders (> 3 AI) cows. Rodrigues et al. Acta Sci Vet 2007;35:1255 (abstract).

P 0.001

NS P 0.001

Significative effect of breeding technique and animal category on CR

P<0.001 but not on PL

(138)

• Heritability for fertility is relatively low : 0.021 to 0.034 (Berry et al. 2014) • Different quantitative trait loci (QTL) are involved in embryonic mortality • (Lefebvre et al. 2011)

• Different lethal mutations affecting six genes, including FANCI gene can influence in utero development in cattle (Valour 2015)

• The 1/29 Robertsonian chromosomal translocation has been described in several beef breeds (but not in the Holstein breed) (Gustavsson 1979)

• In the Holstein breed, two major recessive the deficiency of uridine

monophosphate synthase (DUMPS) can affect embryo survival (Robinson

et al. 1984),

• There is a positive correlation between inbreeding and EM (Berry et al. 2014)

(139)

Overall embryo survival rates would appear to be largely similar in heifers, beef cows and low- to moderate-producing dairy cows, but embryo survival rate is lower in high-producing dairy cows.

Breeding heifers at 26 months of age or older resulted in a 13% reduction in pregnancies per AI, (Kuhn et al. 2006)

(140)

• Negative effect of NEFA (synthetized under NEB, stress or pain) on • oocyte development (Van Hoeck et al. Reproduction 2013,145,33;

Leroy et al. Reproduction 2005, 130,485).

• oviduct cell function Jordaens et al. Theriogenology 2015,84,899) • and subsequent embryo development (Van Hoeck et al ? Plos One

2011,6,e23183).

• Negative energy balance altered gene expression in granulosa cells of dairy cows at 60 days postpartum (Girard et al. Theriogenology 2015,84,1350). • Many fatty acids are different in follicular fluid of heifers and cows

((palmitic acid, stearic acid) in FF from cows and higher docosahexaenoic acid levels in FF from heifers (Butler Reprod Fert Dev 2013,26,1)

Metabolism and risk of EM (From Leroy et al. Reproduction, Fertility and Development 2015,27, 693–703)

(141)

• Many differences in aminoacid composition of follicular fluid between cows lactating and non lactating or heifers (Forde et al. Reprod Fert Dev 2015) • Both fatty acid and amino acids composition are influenced by genotype

(Moore et al. Reprod Fertil Dev 2015)

• The difference in composition can partly explain the differences in

blastocyst development (Matoba et al. Reprod Fertil Dev 2014;26:337–45). • Cows loosing body condition score during the first 3 weeks of lactation had

a lower percentage of pregnancies after an ovulation synchronisation strategy. Such cows have a markedly lower number of viable and

transferable good quality embryos after superovulation treatment (Carvalho et al. J. Dairy Sci. 2014, 97, 3666–3683)

(142)

• b-carotene supplementation in NEB cows was able to increase b-carotene availability in the microenvironment of the maturing oocyte, regardless of energy status (De Bie et al. Adv.Anim. Biosci. 2014, 5, 247–255).

• Better developmental competence after the addition of antioxidants to fatty acid-exposed embryos during culture (Rooke et al. Reprod. Fertil. Dev.

2012, 24, 309–316).

(143)

• _{Experiment conducted in Brazil during the dry season (April to September : 470 mm} of precipitation and and low grass availability) and the rainy season (October to March : 1930 mm and high grass availability).

• _{Cross-bred (Bos taurus x Bos indicus : 328 to 426 kgs) recipients (1,6 animal /ha} during the rainy season and 1,1/ha during the dry season)

• 484 embryos produced in vitro

• _{No significant effect of season 42.3 (rainy) vs. 45.8 (dry) %,}

(144)

144

The use of sexed semen and frozen embryos decreases the pregnancy rate

- _{The PR was higher for embryos grade 1 than grade 3 (P<0.001)} - _{PR was lower after transfer of morulas}

- _{PR was higher after transfer on heifers (OR 1.18)}

(145)

10 11 12 13 14 15 16 17 18 19 20 21 0

The progesterone has a key role in the decrease of EM at three moments

2. At AI : Low level (<0.3 – 0.5 ng)

3. After AI : rapid

increase and high level 1. Before AI : high level

- Better follicular growth - Better quality of oocyte - Reduction of LH pulses

- Better LH surge

- Better maturation of oocyte - Better sperm transport - (Wiltbank et al. 2014)

- Better elongation process - Higher size of embryo at d17

(Carter et al. 2008, Clemente et al. 2009)

- Better synthesis of interferon (Mann et al. 2006, Clemente et al. 2009) and so reduction of the oxytocin endometrial receptors and reduces the pulsatility of PGF2a (Spencer et al. 2007)

Positive effect to synchronize œstrus with CIDR/PRID

(146)

• The risk of embryonic mortality increased with the level of milk production because the catabolism of progesterone is higher in the high milk producing cows (> 40 liters)

(Sangsritavong et al. 2002)

• The risk of embryonic mortality beween days 28 to 34 of pregnancy is decreased in the cows with an additional corpus luteum (1,7 % vs 9,9 %) (Lopez-Gatius 2002, 2004, 2006, Bech-Sabat et al. 2008)

• Higher is the concentration of progesterone 5 days after AI, better is the pregnancy rate (Starbuck et al. 2001)

• During the metoestrus (< 6 days), 60 to 85 % of cows have a too low concentration or a slower increase of the P4 concentration (Stronge et al. 2005)

• Cows with high genetic merit for fertility have 34 % more P4 (Cummins et al. 2012)

• Dairy cows have less or a slower increase of P4 than the heifers (Sartori et al. 2004)

• The use of P4 into synchronization protocols can contributes to increase the pregnancy rate (Bisinotto et al. 2013; Stevenson et al. JDS 2007)

• The increase of progesterone before AI could have a carry-over positive effect during the period of implantation (Cunha et al. 2008).

(147)

P4 contributes to the

elongation process

of the embryonic vesicule

(Carter et al. 2008)

(148)

Reproductive outcomes for for low-yielding dairy (typical of pasture-based systems) cows and heifers compared with high-yielding (typical of intensive grain or total mixed ration-based systems) dairy cows (Diskin et al. 2016. Reproduction, Fertility and Development, 2016, 28, 83–93

dairy cows (typical of intensive grain or total mixed ration-based systems) dairy cows

(149)

The pregnancy diagnosis done by manual palpation don’t increase the risk of EM (Romano et al. 2007)

(150)

10. Which treatments to prevent

embryonic mortality in the cow ?

(151)

• By given

P4

• By increasing the synthesis of P4 (by the CL present on the ovary or by

inducing the development of an additional CL) :

GnRH, hCG

• By reducing the PGF2a effect :

Anti-inflammatory non steroidal

compounds (meloxicam, flunixine meglumine)

(152)

• Early embryonic period

• Administration of P4 during the first two weeks of pregnancy increase the pregnancy rate : + 10 % if before day 6 and + 19 % if after day 6 (P<0.001) (Ball and Lamming 1999)

• Late embryonic period

• Administration of P4 (PRID 1,55 g of P4 for 28 d) beginning between days 36 and 42 of pregnancy reduces the % of embryonic mortality (5,3 % (29/549) vs 12 %

(66/549) (Lopez-Gatius 2004.

• Same results if the treatment begins at day 28 : 6 % (6/102) vs 16 % (16/97) (Bech-Sabat et al. 2007)

• Same results if the treatment begins between day 28 to 35 : 15 % (19/126) vs 21 % (36/172) (Alnimer et Lubbadeh 2008)

(153)

Mann GE, Lamming GE. The influence of

progesterone

during early pregnancy

in cattle. Reprod.Domest.Anim.

(154)

A IVP4 deliver 90 mg of P4 per day (Rathbone et al. J Contrl Release 2002)

So the P4 concentration increase of 1 ng / d (Cerri et al. 2009 Anim Reprod Sci 2009)

Treated

Control

(155)

• The injection 2 days after oestrus increases the surface of the CL (Maillo et al. 2013)

• The injection (1000 to 3300 UI) 4 to 7 days after oestrus induces the increase of P4 into 3 days.

• The injection 5 days after AI increases by 3,5 % (37,3 vs 40,8 %) the pregnancy rate (mainly in primiparous cows (49,7 vs 39,5 %) than in

multiparous cows (35,7 vs 36 %) (Nascimiento et al. 2013 : a metaanalysis of 10 studies).

• Injection betwen days 5 to 7 induces the ovulation of the 1st wave dominant follicle and increase the probability to have an accessory CL . So the

pregnancy rate is increased (45,8 vs 38,7 %) (Santos et al. 2001)

(156)

1 5 6

S increase NS increase

NS decrease

Effect on pregnancy rate of a

GnRH injection

11 to 14 days after AI

A meta-analysis of 19 studies (10.945 cows) (Peters et al. 2000)

(157)

Antiinflammatory non steroidal drugs (AINSD)

Meloxicam Half-life : 35 h Flunixine Meglumine Decrease of PGFM for 12 h Carprofen Half-life : 44 to 64 h Ibuprofen

(158)

(159)

Publications related to the use of NSAI drugs

at embryo transfer or after AI : General data

(160)

Effect on the pregnancy rate (%) of some NSAI drugs

used

at the time of embryo transfer

in the cow

(161)

Effect on the pregnancy rate (%) of some NSAI drugs

used

13 to 16 days after AI

in the cow

(162)

• On general the use of NSAI increases the pregnancy rates but the difference is not always significative

•

• The effect seems to depend on the quality of the embryo : (better effect if the quality of embryo is higher (grade 1 vs grade 2) (Lopes et al. 2015)

• The negative effect has been reported. It could be due to the long acting effect of MEL and the inhibition of the positive effect of PGF on implantation process (Erdem and Guzeloglu 2010).

• Several authors report differences between locations (nutrition or management effect ?)

Some conclusions and observations about the use of NSAI drugs at

embryo transfer or after AI

(163)

• The effect increases with the stress of animals (chute vs blocks head) or transportations by truck (4 to 6 h)after AI (Geary et al. 2014)

• The effect increases with the level of « exitability of animals (Kasimanickam et al. 2018)

• In the « excited » group (fast chute exit and jump) : Increase of the PR (56,8 %) compared to non treated (46,3 %) (P<0.05)

• In the « calm » group (slow chute exit and walk) no difference between treated (59,3 %) and non treated (59,4 %) (P=0.15)

Some conclusions and observations about the use of NSAI drugs at

embryo transfer or after AI

(164)

• The effect depends on the quality of embryo and method of freezing (Schrick et al. 2001) • Grade 1 : 66 vs 50 (P<0.001) • Grade 2 : 65 vs 54 (P<0.09) • Grade 3 :50 vs 47 % (P=0.8) • Morula : 65 vs 51 (P<0.002)

• Embryo frozen with ethylene glycol 70 vs 47 % (P<0.05) • Embryo fresh or frozen with glycerol : no effect