Minggu, 26 Juni 2011

Tips mempercepat proses kawin burung kenari

Anda sedang menangkarkan kenari? Kesulitan dalam proses perkawinannya? Atau tidak juga mau kawin meski jantan betina sudah sama-sama terlihat birahi? Jangan bingung, nih ada beberapa tips  dari sejumlah penangkaran burung, terutama penangkaran Mas Kuwat di kawasan Klewer Sukoharjo.
Syaratnya utamanya mudah, yakni selain kita punya kenari jantan yang siap kelamin dan akan kita jadikan pejantan, kita juga harus punya kenari jantan lainnya yang juga sudah gacor.
Tips dan langkah-langkah mengawinkan kenari
1. Tidak pernah mencampur jantan betina dalam waktu lama. Artinya, selama penjodohan hingga mau kawin, jangan pernah dicampur dalam satu sangkar.
2. Ketika kenari jantan sudah benar-benar gacor dalam kesehariannya, dan betina sudah matang kelamin (kloaka terlihat bengkak memerah) dan sudah menyusun sarang di sangkar lainnya, proses mengawinkan baru dimulai.
3. Jika kondisi sudah seperti yang saya sebutkan di atas, maka pilihlah waktu pagi hari setelah kita selesai bersih-bersih dan memberi pakan semua momongan, masukkan jantan kenari yang sudah siap itu ke sangkar kenari betina.
4. Jika setelah dimasukkan si kenari jantan tidak juga segera mau naik mengawini si betina, ambillah kenari gacor kita yang lainnya, dan dekatkan ke sangkar jantan-betina yang sedang akan kita kawinkan.
5. Begitu melihat pesaing datang, biasanya kenari jantan akan segera “naik” ke atas betina dan terjadilah perkawinan.
6. Biarkan jantan dan betina kenari berduaan sampai sekitar 1 jam. Dalam rentang waktu itu, biasanya terjadi 2-3 kali perkawinan atau lebih.
7. Setelah poin 6 terlampaui, pisahkan jantan dan betina kenari sehingga keduanya tidak saling lihat.
8. Ulangi kesemua langkah di atas selama 3 hari berikutnya. Nah, setelah lewat 4 hari masa perkawinan, kenari jantan dan betina tidak perlu dicampur lagi. Betina akan bertelur dan mengerami sendiri telur-telurnya.
Tips dan info lain:
1. Jika kita mengawinkan kenari jantan dan betina pada pagi hari, maka setelah sekitar 6 jam atau sore harinya, si jantan bisa untuk mengawini kenari betina yang lain, dengan proses sama dengan tips yang saya sebutkan di atas.
2. Jika kenari jantan tidak juga segera gacor merayu betina meski secara umum terlihat sehat atau kenari betina tidak juga matang kelamin meski sudah berusia di atas 7 bulan; atau telor-telor kenari tidak isi dan karenanya tidak bisa menetas, kita perlu memastikan bahwa si jantan bisa memproduksi sperma yang “berisi” dan kesehatan reproduksi betina benar-benar maksimal.Selama kondisi alat-alat reproduksi dalam keadaan normal, Bird Mature sudah terbukti meningkatkan kesempurnaan proses reproduksi burung-burung penangkaran. Tidak hanya kenari, tetapi semua jenis burung.
3. Jika burung-burung kenari anakan kita gampang mati, atau kakinya sering pengkor, lembek, karena daya tahan tubuh secara umum lemah, kita perlu memastikan bahwa indukannya mengonsumsi mineral
Bird Mineral tidak hanya bagus untuk anakan tetapi juga indukan karena Bird Mineral menjadikan bulu kuat, mulus, berkilau sehabis molting atau ngurak alias mabung; burung tidak terkena rachitis (tulang-tulang lembek, bengkok dan abnormal); bebas paralysa (lumpuh); bebas perosis (tumit bengkak); menjadikan anak burung menetas sehat; burung tidak mengalami urat keting (tendo); burung tidak terlepas sendinya, tidak tercerai (luxatio); paruh tidak meleset, tidak kekurangan darah sehingga pucat dan lemah; burung di penangkaran bisa segera bertelur, telur berisi, produktivitas tinggi, daya tetas tinggi; kematian embrio rendah.

Membedakan jenis kelamin lovebird

Membedakan jenis kelamin lovebird termasuk pekerjaan gampang-gampang susah. Gampang untuk jenis-jenis tertentu tetapi susah untuk jenis lainnya, apalagi kalau masih anakan. Untuk membedakan jenis kelamin lovebird bisa digunakan cara sederhana sampai yang ilmiah.
Berikut ini adalah serba-serbi mengani perbendaan lovebird jantan dan lovebird betina yang saya ambil dari tulisan Siti Nuramaliati Prijono dalam buku berjudul Lovebird terbitan Penebar Swadaya.
A. Berdasarkan penampilan luar.
Menurut Siti Nuramaliati, berdasar tingkat kesulitan untuk membedakan jenis kelamin lovebird (dan burung secara umum) maka dapat dibedakan 3 kelompok lovebird. Ketiga kelompok tersebut adalah kelompok dimorfik (jenis kelaminnya sangat jelas dapar dibedakan), kelompok intermediate (jenis kelaminnya agak sulit dibedakan dari penampilan burung), dan kelompok lovebird kacamata (perbedaan jenis kelaminnya tidak konsisten). Namun secara umum pada banyak jenis lovebird relatif mudah dibedakan jenis kelaminnya dengan melihat pada penampilan luarnya.
a. Kelompok lovebird dimorfik
Beberapa jenis lovebird yang termasuk dalam kelompok dimorfik di antaranya lovebird abisinia, lovebird madagaskar dan lovebird muka merah.
1. Lovebird abisinia (Agapornis taranta)
- Lovebird jantan berat badan 65 gram, dahi berwarna merah.
- Lovebird betina berat badan 55 gram, dahi berwarna hijau.
2. Lovebird madagaskar (Agapornis cana)
- Tidak ada perbedaan berat badan antara lovebird jantan dan lovebird betina.
- Lovebird jantan kepala dan leher berwarna abu-abu
- Lovebird betina bulu tubuh keseluruhannya berwarna hijau
3. Lovebird muka merah (Agapornis pullaria)
- Lovebird jantan: dahi dan muka berwarna merah-oranye, tunggir (bulu di atas pantat, di bawah ujung lipatan sayap) berwarna biru muda, bulu terbang dan bagian bawah bulu sayap berwarna hitam.
- Lovebird betina dahi dan muka lebih didominasi warna oranye dibandingkan warna merah, bagian bulu penutup sayap berwarna hijau dan di tepi sayap berwarna kekuningan.
b. Kelompok intermediate
Dua jenis lovebird yang termasuk dalam kelompok intermediate adalah lovebird black collared dan lovebird muka salem.
1. Lovebird black collared (Agapornis swinderniana)
Lovebird jantan dan betina sangat sulit dibedakan dan tampak serupa dalam penampilan luarnya.
2. Lovebird jantan dan betina serupa dalam penampilannya, meskipun pada umumnya lovebird betina mempunyai bulu di bagian kepala dengan warna yang lebih pucat.
c. Kelompok lovebird kacamata
Empat jenis lovebird yang termasuk dalam kelompok lovebird kacamata adalah lovebird nyasa (Agapornis lilianae), lovebird pipi hitam (Agapornis nigrigenis), lovebird topeng (Agapornis personata), lovebird fischer (Agapornis ficheri).
Keempat jenis lovebird ini sangat sulit dibedakan antara jantan dan betina. Meskipun demikian ada sedikit perbedaan berat badan antara jantan dan betinanya. Satu keunikan dari lovebird kelompok kacamata adalah pada saat menjelang musim berkembangbiak burung betina akan membawa bahan sarang di bawah bulu tunggir dan bulu punggung bagian bawah.
B. Membedakan jenis kelamin tidak berdasarkan penampilan luar.
Pada jenis lovebird yang tidak dapat dibedakan jenis kelaminnya berdasarkan penampilan luarnya yang spesifik maka akan sulit untuk membedakan lovebird jantan dan lovebird betina. Pada kejadian ini makan ada beberapa cara untuk digunakan memnedakan lovebird jantan dan lovebird betina.
a. Bentuk tubuh.
Lovebird betina cenderung memiliki tubuh yang kekar dan lebih berat. Namun kriteria ini tidak mutlak sifatnya.
b. Warna
Lovebird jantan mempunyai warna yang lebuh terang dari lovebird betina. Meskipun demikian hal itu tidak selalu benar karena warna bulu juga tergantung pada makanan, iklim, dan variasi geografis.
c. Cara bertengger
Lovebird betina bertengger dengan jarak antarkaki lebih lebar dibandingkan lovebird jantan.
Perbedan jantan dan betina dari bukaan kaki
d. Bentuk ekor
Lovebird betina mempunyai ekor dengan bentuk lebih rata dibandingkan pada ekor lovebird jantan yang berbentuk agak meruncing.
Perbedan jantan dan betina dari bentuk ekor
e. Membangun sarang
Kegiatan membangung sarang lebih intensif dilakukan oleh lovebird betina ketimbang jantan. Lovebird menggigit-gigit di luar sarang pada cabang-cabang dan batang yang lebih tebal. Lovebird betina akan megambil kulit kayu dan mengumpulkannya untuk membuat sarang, sedangkan lovebird jantan menyuapi lovebird betina. Namu hal ini juga tidak mutlak karena ada lovebird jantan yang juga aktif mengumpulkan bahan sarang.
f. Perabaan pada tulang pubis (supit urang).
Lovebird memiliki dua tulang pubis (supit urang) pada bagian pinggulnya. Pada musim berkembang biak, tulang pubis lovebird betina menjadi lebih elastic dan jarak antara kedua tulang pubis tersebut melebar karena pengaruh hormone. Keadaan tersebut dapat dirasakan dengan rabaan tangan. Pada lovebird jantan, jarak antara dua tulang pubis tersebut sempit. Teknik perabaan ini hanya dapat digunakan bila kegiatan seksual lovebird betina dengan aktif.
g. Pemeriksaan dengan alat laparoscopy
Untuk mengetahui jenis kelamin lovebird juga bisa dilakukan dengan menggunakan alat laparoscopy. Lovebird yang akan diperiksa jenis kelaminnya harus dibius dulu. Setelah itu dilakukan operasi kecil pada bagian kiri tubuh burung di antara tulang rusuk, tulang pinggang dan tulang paha. Dari bagian yang dioperasi itu dimasukkan alat laparoscopy untuk melihat ada tidaknya ovary (indung telur). Jika ada ovari maka lovebird tersebut dipastikan betina. Cara ini hanya bisa dilakukan jika burung sudah dewasa.
h. Pemeriksaan DNA
Cara lain untuk mengetahui jenis kelamin lovebird adalah dengan menguji DNA yang dapat diperoleh dari darah atau bulu burung. Setelah DNA diekstrak dengan larutan tertentu dan proses lebih lanjut, lalu hasilnya dipotret dengan Polaroid. Apabila dalam foto tersebut terlihat dua pita maka lovebird tersebut dapat dipastikan berkelamin betina. Namun jika terlihat hanya satu pita, lovebird itu bias dipastikan jantan.
Cara ini dianggap lebih cepat dan hasilnya lebih akurat. Namun biaya uji DNA sangat mahal. Selain itu di Indonesia belum banyak laboratorium yang menawarkan jasanyan untuk memeriksa jenis kelamin burung dengan uji DNA.
Pasangan sejenis juga bercumbu
Pada jenis lovebird yang tidak dapat dibedakan antara jantan dan betinanya berdasarkan bentuk tubuh dan warna bulunya sering terjadi kesulitan untuk memperoleh pasangan yang sesuai,.
Sering terlihat dua ekor jantan berperilaku seperti pasangan lovebird yang berlainan jenis. Hal yang sama juga terjadi pada dua lovebird betina. Bahkan pada pasangan lovebird betina ini apabila bertelur maka jumlah telurnya akan lebih banyak dari pasangan yang normal, tetapi telur tersebut tidak fertile alias tidak akan menetas jika dierami.
Hal yang membedakan antara pasangan jantan-jantan dan betina-betina adalah pada pasangan jantan-jantan tidak akan membuat sarang karena perilaku itu hanya milik lovebird bertina.
Ada yang menyatakan bahwa lovebird jantan adalah yang menyuapi pasangannya sedangkan betina yang disuapi. Tetapi hal ini tidak benar karena lovebird betina juga sering menyuapi lovebird jantan untuk menarik perhatian si jantan.
Juga tidak benar bahwa lovebird betina memiliki paruh dan kepala yang lebih kecil ketimbang lovebird jantan. Dan tidak tentu benar bahwa lovebird jantan memiliki kepala yang lebih lebar dengan paruh yang lebih runcing. (*)
Semoga bermanfaat.

Membedakan Jenis Kelamin Kenari II

Banyak para hobies bingung memilih kenari bakalan yang akan di beli, sangat sulitmembedakan dilihat dari postur tubuh bahkan ada yang langsung beli begitu melihat bodiyang besar dan tegap setelah sampai dirumah dan dipelihara kira-kira 3 bulan barumereka bingung, kok burung kenari saya gak bunyi-bunyi mas. komplainlah mereka ke penjual burung tersebut.itu sedikit pengalaman saya pada saat kongkow di penjual Kenari di Pasar Burung Prambanan.Saya akan coba ulas sedikit mengenai cara membedakan kenari Jantan dan Betina atau bahasa kerennya Sexing.
Ciri jantan dan betina
 Meskipun menentukan jenis kelamin kenari bukan hal yang mudah dan mereka yangsudah berpengalaman pun banyak menemui kesulitan, tetapi ini ada tips yang bisadigunakan (tetapi bukan hal yang mutlak): 

1.Sejak usia 3 hari pembuluh vena di sebelah perut akan turun menuju “ vent( lubang angin / dubur ) dan akan menuju bagian depan dari “ vee “ yang ada dibagian depan “vent ,ini nampak pada burung kenari jantan yang masih muda.Tapi pembuluh vena ini tidak akan tampak pada kenari betina. Walaupun pembuluh ini ada di seluruh kenari jantan , akan lebih baik jika menganalisanya sebelum kenari jantan tersebut tumbuh bulu. 

2.Pada 5-8 hari ,ambil semua anak-anak kenari yang terdapat pada sarang yang sama (satu indukan) taruh ke handuk dan sejajarkan seperti posisi kuda balap yang ada pada pacuan kuda. Yang melompat paling jauh adalah kenari jantan. 

3.Burung- burung tersebut jika diperhatikan mempunyai pusat kaki bawah yang panjang sementara yang lainnya punya 3 telapak kaki yang memiliki panjang yang sama. Salah satu yang memiliki pusat kaki yang panjang akan sulit untuk  bergabung dengan yang lain dan ini adalah kenari jantan. Kenari betina lebih mudah untuk bergabung karena ketiga telapak kakinya memiliki ukuran yang sama.

 4.Lihatlah bagian tulang belakang mereka. Di sepanjang tulang belakang kenari jantan warnanya lebih kaku dan lebih pekat . Untuk yang betina warnanya kaku dan pekatnya tidak sampai sepanjang tulang belakang. Dengan cara itu bisa diidentifikasi.

 5.Sebelum anakan-anakan kenari tersebut berbulu. Liatlah ke bagian kepala mereka.Untuk yang jantan lebih pipih (datar).Untuk yang betina lebih bulat. 

6.Sekitar usia 6 – 7 hari anak2 kenari tersebut mulai membuka mata. Jika jantan maka matanya sejajar dengan paruhnya. Untuk yang betina, letak mata diatas paruh yang menyebabkan tampilan kepalanya lebih bundar.
 
7.Ketika anak2 kenari diberi makan disarangnya oleh induknya .perhatikan tingkah laku anak2 tersebut. Yang berdiri paling tinggi , teriak paling keras dan yang paling ingin diberi makan duluan itu berarti yang jantan. Yang betina kakinyalebih pendek makanya mereka tidak berdiri tinggi dan karenanya mereka diberimakan lebih sedikit dibanding yang jantan.

 8.Usia 28- 30 hari beberapa kenari2 muda terlihat mempraktekkan suara mereka untuk berkicau dengan baik. Bukan yang paling awal berkicau itu yang terlihat paling baik, burung 2 tersebut hanya suka berlatih berkicau lebih awal saja. 

9.Sekitar 5 bulan atau 5 bulan lebih 2 minggu, kenari jantan bisanya warnanya lebih terang. pada bagian tertentu warnanya lebih tajam khususnya dibagian dekat kepala. Pusat kepala pada burung kenari betina yang berwarna kuning akan menjadi sangat terang

 10.Seandainya usia 6 bulan belum ketahuan jenis kalaminnya ,kenari2 yangdicampur diantara kenari2 maka. Kenari betina akan duduk lebih rendah lebihdikaren akan struktur badannya. Dan juga biasanya kenari betina akan berkelahi dengan kenari jantan ketika ingin berkicau atau mempraktekan suaranya.Meskipun sang betina dalam kondisi tidak sedang dalam masa bertelur dan inihanya sekedar ingin berkicau saja..

Rabu, 15 Juni 2011

BOVINE EMBRYO TRANSFER


1.    Superovulation of Donor Cows

The cow usually ovulates only one egg during each estrous cycle.   Many years ago, it was found that the administration of hormones with FSH-like activity would stimulate extensive development of mature follicles in the ovaries and thus allow for ovulation of a larger number of eggs.   The most commonly used hormone preparations for inducing such superovulatory responses are pituitary extracts from swine or sheep containing FSH, and pregnant mare’s serum gonadotropin (PMSG), a hormone with FSH-like activity that is found in the serum of pregnant mares.
Administration of the superovulating hormone should begin 4 to 9 days prior to the expected onset of estrus.  When using PMSG, a single injection will suffice. FSH should be given twice daily during the entire period of superovulation.   In most cases, prostaglandin F2a (PGF) is used in conjunction with superovulation, so that one can accurately predict the time of the onset of estrus.  A typical schedule to superovulate a cow would be to begin injections of FSH 9 to 14 days after the last estrous period and to inject PGF on the fourth day of FSH treatment.  The cow would be expected to be in estrus 36 to 60 hours after the injection of PGF.
Superovulation does have some problems, the largest of which is the wide variation in response.  Thus, some cow will not superovulate at all.  The optimal response, in terms of the number of transferable embryos recovered, appears to be in the order of 10 to 15 ovulations.




Table 1.   Bovine Superovulation Protocol

Day
0. Estrus

Dairy breeds
Dual purpose breed
10



11


12


13



14


15


22
a.m.*
p.m.


a.m.
p.m.

a.m.
p.m.

a.m.

p.m.

a.m.
p.m.

a.m.
p.m.
-
2.500 - 3.000
PMSG

-
-

-
-

Prostaglandin

-

-
-

-
AI + Anti-PMSG Antiserum
-
7 mg FSH


7 mg FSH
6 mg FSH

6 mg FSH
5 mg FSH

5 mg FSH +
Prostaglandin
4 mg FSH

4 mg FSH
-

-
AI + HCG 3.000

Embryo Collection
-
8 mg FSH


8 mg FSH
7 mg FSH

7 mg FSH
5 mg FSH

5 mg FSH +
Prostaglandin
5 mg FSH

5 mg FSH
-

-
AI + HCG 3.000

     * Superovulation can be commenced between days 9 and 15

 

2.    Nonsurgical Recovery of Bovine Oocytes


Bovine ovum recovery attempts are made from 6 to 8 days (D 6 – 8) after estrus.  Most ova have entered the tip of the uterine horn at this time and still have their shell (zona pellucida) intact (which is believed an advantage when recovering and transferring embryos).   Donors are starved and deprived of water for 24 hours prior to ovum recovery, except for lactating dairy cows.   Rectal palpation and manipulation (of instruments) are facilitated in a partially starved animal.
Beef cows are usually placed in a squeeze chute and dairy cows in a head bail before preparation.  The tail-head is clipped, washed with soap and water, and sterilized using an iodine compound followed by alcohol.  A suitable site (between 2 articulating vertebrae in the sterilized area) (intercoccygeal groove 1 – 2) is selected for injecting 5 to 6 ml of a 2% local anaesthetic (Xylocaine HCl, Lidocaine HCl, Procain HCl etc) around the spinal cord (Epidural anaesthetic).   The epidural injection prevents the cow from straining on your arm when it is in the rectum palpating the reproductive tract and manipulating instruments.   In some older, particularly fat cows, it is difficult to obtain a satisfactory epidural block.  If two attempts are unsuccessful, it is recommended to apply 40 mg of an acepromazine intravenously for general relaxation (as tranquilizer).   The tail is tied out of the way, and the rectum may be emptied of faeces.
The vulva area is washed with soap and water and sterilized with an iodine compound and alcohol.   With the arm in the rectum, ovaries are located, and follicles and corpora lutea are estimated to give an approximate idea of the number of ova released.   The vulvar lips are parted, and a plastic pipette is introduced into the vagina and placed in the lumen of the cervix.   Under gentle pressure, the pipette is manipulated just through the cervix and then, under vacuum by a 20 cc syringe, slowly pulled backward to aspirate and remove the mucus from the cervix.
A 16 or 18 G (French gauge), sterile, 2-way Foley catheter (depending on the size of the uterus) with a mandarin stiletto of stainless steel inside is gently inserted into the vagina and on into the cervix.  It is then manipulated into the selected horn so that the inflatable balloon is situated clearly behind the palpable bifurcation of the two horns.  The balloon is gently inflated with a 10 to 15 ml of air, depending on the size of the uterine horn.  The balloon should be tight so that medium cannot leak out between the balloon and wall of the uterus.  However, if the balloon is too tight, the endometrium will rupture and bleeding occurs, which will adversely affect ovum searching.  After the catheter is placed in its final position with the balloon inflated, the mandarin stiletto is removed.
Total of 1.000 ml flushing medium warmed to 37 °C is prepared in a water bath. A Y-junction tubing is connected to the bag with flushing medium and attached to the tube of the flushing catheter.  The medium is then flushed slowly into the lumen of the chosen uterine horn (500 ml flushing medium for each horn).   Under gentle massage of the uterus wall via rectum the medium is collected through the outlet tubing of the Y-junction and into a filter.
The catheter can be changed to the opposite horn by either removing it completely from the uterus and replacing it, or the catheter can be pulled back into the body of the uterus after deflating the balloon.  Then the mandrin is inserted into the lumen of the catheter until it reaches the tip, which can be palpated.  After placing the catheter, the process of flushing is repeated with the second horn.  The filter filled with flushed medium is taken to the laboratory. The Petri dish is placed for searching the ova under a stereo microscope with a transluminant base at a magnification rate of 10-12 times.
                        

Fig. 1: BoviPro™ flushing medium, Y-junction tubing and Miniflush™ Filter


3.    Classification of Bovine Embryos


Location, identification, manipulation and classification of ova and embryos require experience and will only be learned by considerable practice.  Ova are easily missed, so each dish should be searched twice by one person and then checked by a second one.
Ova are manipulated with small glass pipettes or capillary tubes, or 0.25 ml straws attached to some suction device.   They are retrieved from the medium in about 0.1 ml of fluid and placed in small 5-well dishes with culture medium.   When the searching is completed, the ova are classified, based on their morphological appearance at a magnification rate of 40-50 times and washed by being passed through three sterile solutions of holding medium.
The embryos are then stored at room temperature until transfer or freezing. Embryos will continue growing in the dish and, for example, those collected seven days after estrus will change gradually from the morula stage to early blastocysts, which is a good indication of the viability of the embryos and the efficiency of the medium used.   In the first 12 hours of culturing at room temperature, very little viability is lost, then, up to 24 hours a little decrease occurs, after 24 hours there is a rapid loss.
After superovulation, the average fertile donor will yield in total 10 ova, fertilized, unfertilized and or degenerated.   Usually 5 to 7 embryos are of transferable quality.


a.      Identification of the embryo

The bovine embryo is about 160 μm (0.16 mm) in diameter through day 8 of development.  This size is beyond the resolving power of the naked eye. Therefore, a stereo microscope (magnification, 10-50 times) is used to identify embryos in uterine flushing.

                                                          Fig 2.  Stereo microscope

The morphology of all bovine embryos is similar through day 9 of development, and it is composed of a cellular mass surrounded by an acellular matrix known as the zona pellucida.  The area between the embryonic mass and the zona pellucida is known as the perivitelline space.  Cells composing the embryo are termed blastomeres.  During embryonic development blastomere number increases in a geometric fashion through the 16 cell stage (e.g. 1 – 2 – 4 – 8 – 16 cells).  After the 16 cell stage, cell divisions become asynchronous and individual blastomeres become difficult to distinguish.  At the 40 to 60 cell stage, the bovine embryo undergoes compaction; that is, the blastomeres loose their spherical shape and begin to adhere to one another.  Embryos at this stage of development are termed compacted morula and are most often recovered on day 5 to 7 after standing estrus.  During the late morula stage, blastomeres begin to differentiate giving rise to two cell types trophoblast and inner cell mass.  These two cell types become readily distinguishable at the blastocyst stage.  A blastocyst is a layer of trophoblast cells surrounding a cavity called the blastocoele.  The cells of the blastocyst are specialized since the inner cell mass will form the fetus proper and the trophoblast will form the placenta.  Blastocysts are found most often 7 to 10 days after estrus.
Problems may be encountered with blastocysts collected after day 8 because the zona pellucida is lost between day 8 and 10.  When zona-free blastocycts are collected, a greater risk of damage due to handling is encountered.  Additionally, zona-free blastocysts are “sticky” and may adhere to tubing and glassware.
Identification of embryos in uterine flushings is based on several of the above mentioned morphological features.  First, the zona pellucida is used as a landmark. The zona, being a translucent structure present on all embryos through day 9, clearly distinguishes embryos from other cellular debris in flushing.  Second, the color of the embryo (a dark amber) facilitates identification because it is usually darker than other uterine debris.  Third, the embryo is spherical and tends to roll along the bottom surface of the searching dish.  Finally, knowing the stage of the embryo to be collected, from the day estrus, speeds identification of embryos in the collection medium.

b.     Classification of the embryo

To date there is no single parameter of embryo morphology which correlates with pregnancy rate, except that unfertilized ova will not result in calves.  Therefore, several characteristics or recovered embryos are assessed before transfer.  Recovered embryos are classified from excellent to poor based on their gross morphological appearance.
-          Excellent embryos have no visible imperfections.
-          Good embryos show a single small imperfection such as an extruded blastomere, asymmetrical shape, or slightly retarded development compared to other embryos in the same flush.
-          Fair embryos possess more than one of the above imperfections and are usually 1 to 2 days retarded in development.
-          Poor embryos show signs of cellular degeneration, little or no cellular organization and are 2 to 3 days retarded in development.
-          Several degenerated embryos are more than 3 days retarded in development.
-          Unfertilized ova do not show any sign of development.

After embryos have been classified as to quality, individual imperfections are recorded on an embryo description sheet.  The age of the embryo, from estrus to collection is recorded, as it is an estimate of the number of cells present in the embryo. Compactness of the blastomeres is estimated; loosely arranged blastomeres may indicate poor development or degeneration.  Very dark or light blastomeres may indicate that degenerative processes have occurred.   The cellular mass of the embryo should be spherical and symmetrical.  Large vesicles in blastomeres are indicative of some degree of degeneration.  Blastomeres may be extruded during any stage of development.  A small number of extruded blastomeres is usually not detrimental to further embryonic development, but a large number (over 5) of extruded blastomeres indicate that the embryo may be undergoing more degenerative than development processes.  The proportion of the perivitelline space occupied by the embryo is also recorded.  Generally, the percentage of perivitelline space occupied by the embryo is least at the compact morula stage.  Finally, the stage of the embryo is recorded, for example morula, blastocyst, etc.

Normally developed embryos:

                
Early morula                                       Compacting morula                    Beginning blastocyst
      
Beginning blastocyst                      Blastocyst

Fig 3.  Bovine normal embryos



Partially degenerated embryos:

                
16-cell-stadium (retarded)         Degenerated blastomere              Shrunken morula                                                                                               
Morula with degenerated blastomeres

Useless embryos:
            
1-cell-stadium                             Empty zona (broken)                     Irregular divisions (unfertilised)                                                                                             

                 Fig 4. Abnormal bovine embryos
Blastomeres falling apart


c.      Handling


Both the physical and chemical environments of the recovered embryos must be maintained close to those of the uterine environment to insure maximum pregnancy rates after transfer. Embryos are fragile and will not tolerate small changes in either environment.
Embryos are often maintained at room temperature for several hours without adverse effects. Caution should be employed since repeated sudden changes in temperature may have adverse effects on embryo viability.
As embryos are identified in the flushing medium, they are immediately transferred to a small petri dish containing fresh sterile (0.20 μm filtered) culture medium (= holding medium). Embryos are tentatively classified as good or bad and recorded so that a quick estimate of embryo number can be made. When all embryos are recovered, they are serially washed through small dishes of sterile culture medium and finally placed into a fresh well containing sterile culture holding medium. Embryos are then for transfer or cryopreservation.


                                   
Fig 5.   5-well culture dish


4.   Nonsurgical Transfer


Recipients should be healthy, mature heifers or young cows in good condition. At least two normal cycles should have been established before use to determine whether they are in natural heat on the same day as the donor.
Recipients are placed in chutes or in head bails. A small epidural anaesthesia of 5ml 2% lidocaine is infused around the spinal cord. It takes about 5 minutes for the drug to take full effect.
Sterile 0,25ml straws and transfer sheaths are prepared. The embryo is sucked up into the straw by attaching a suction device to the end containing the plug. About 0,1ml of holding medium is placed behind the front bubble which helps to wash the embryo into the uterus.








 


Marker
 
Holding Medium
 



The loaded straw is placed in the transfer instrument and covered by a sheath. With the hand in the rectum, the corpus lutem on the ovary is located. The vulval lips of the recipient are parted by an assistant, and the transfer instrument is placed in the vagina without touching the vulval lips or the first part of the vagina. Since most embryos are transferred 6, 7 or 8 days after heat, the cervix is tightly closed, especially in heifers. Therefore, it is usually much more difficult to pass the instrument then than during estrus. Once through the cervix, the device is directed to the selected horn. Remember, the true bifurcation is only about 1cm from the cervix, so as the rod leaves the cervix, it must be immediately inclined toward the required horn. The horn is raised and straightened ahead of the tip of the gun before it is gently pushed up the horn. Once resistance is met, the embryo is expelled and the rod removed. An experienced person averages about 50 seconds to implant the embryo. The problems encountered using this method are:

1)     Placing the transfer instrument through the closed cervix in heifers
2)     Depositing the embryo relatively high up in the uterine horn without causing extensive damage to the delicate lining of the uterus (endometrium)
3)     Considerable natural manual dexterity and experience are required
In fact, pregnancy rates depend upon many events, such as quality of embryos and recipients, the skill of embryo transfer technicians, and the preparation and management of the recipients.


5.   Estrous Synchronization for Bovine Embryo Transfer


Prostaglandin F2a (PG) is efficiently used for embryo transfer programs in both donors and recipients.
Following the recording of regular estrous cycles, donors give a much more consistent response to the gonadotropins.  Usually PG is given 3 or 4 days after the first injection of gonadotropin.  Heat or estrus then occurs, in the superovulated cow only 48 hours later.  Even after 4 superovulation treatments, the response to the PG remains consistent, with the donor averaging 50 hours from injection to heat.  The manufacturer’s recommended dose is 25 mg intramuscularly, however, 35 mg may be used on heavy donors (more than 500 kgs body weight).   The dose of PGF can be used as an intrauterine infusion with only 5 mg of PGF (1/5 dose of intramuscular dose).
Another use for PG treatment on donors is to terminate an unwanted pregnancy. Following superovulation and recovery, an embryo is occasionally not flushed out and the pregnancy continues.  These early pregnancies are efficiently removed by injecting 25 mg of PG. In 72 hours, the pregnant donor comes into heat and aborts the foetus.
Frequently, and especially for on-the-farm embryo transfer, it is an advantage to superovulate 3 to 5 donors at one time. PG can be used successfully between days 6 and 17 of the donor’s estrous cycle. The standard 25 mg dose is sufficient.
Recipients must be in heat either one day before, the same day, or the day after the donor’s heat.  With PG, a relatively small group of recipients can be synchronized into heat the same day as the donor.

There are two regimens which can be used:
1)     Palpate recipients for corpora lutea (CL) and inject 25 mg of PG into those with a CL. Heat occurs 48 to 96 hours later, peaking at 72 hours.
2)     Inject all recipients with PG regardless of the presence or absence of a CL. Repeat the 25 mg injection of PG 11 days later.  Estrous behaviour will again peak between 2 and 3 days.  Those recipients not responding on the first occasion because they were in the first 5 days of the estrous cycle, respond the second time as they are then in the mid-to-late period of the cycle, while those recipients that did respond are in the early to mid-cycle period at the time of the second PG injection.
Since the average donor yields 7 transferable embryos, ten recipients per donor is a reasonable number to prepare, to have eight on the average suitably synchronized with the donor. The recipients must be injected with PG on the same day as the donor for the final synchronization.

6.   Freezing of Bovine Embryos

- Procedure of Freezing Embryos


1)     On day 7 after estrus donor cows are flushed non-surgically for embryo recovery.

2)     Embryos are washed at least three times in holding medium.

3)     2 cryoprotectants are available for freezing embryos:

1. Ethylen glycol, allowing direct transfer after freezing and thawing, is today
    mostly used (BoviPro™ Ethylene Glycol with sucrose, Ref. No. 19982/1302).

2. Glycerol requires stepwise exposure to the embryo before freezing and
    stepwise removal after thawing (BoviPro™ Glycerol with sucrose, Ref. No.
    19982/1303, BoviPro™ Embryo Rehydration Kit, Ref. No. 19982/1404).
    Cryoprotectants function by lowering the amount of intracellular ice at any
    given temperature and may stabilize cellular membranes, thus reducing
    damage during freezing and thawing.

4)     Embryos are suspended in the freezing medium. Straws are loaded with the embryos. In the case of Ethylene Glycol freezing, straws are loaded as described in the picture:

























 


Marker
 
Ethylene Glycol Medium
 

5)     The straws are then placed into a programmable freezer, precooled at -7 °C, working either by vaporizing liquid nitrogen or by a mechanically refrigerated alcohol bath.
6)     After three minutes of equilibration in the -7 °C chamber the straws are “seeded” by local cooling of the straw wall with a forceps which has been precooled in liquid nitrogen. This process causes ice to form in the solution and enables the required slow and uniform cooling rate to be achieved. Ice formation starts the process of cellular dehydration, thus reducing the amount of intracellular ice.
7)     The embryos in the straws are then cooled slowly at 0,3 °C/min to -35 °C.
8)     The straws are then plunged into liquid nitrogen.
The whole freezing process takes about 2 ½ hours and it is speculated that the embryos, once frozen, will have a storage life similar to that of frozen spermatozoa.
Basically, 2 types of freezing machines are available for slow freezing of embryos: alcohol bath freezers and liquid nitrogen freezers are both suitable.

 

Fig 6. Bovine embryo freezing machines


- Procedure of Thawing


According to the two different types of cryoprotectants the embryos are thawed as follows:
a)     Ethylen glycol: No special thawing procedure is required.
b)     Glycerol:
-          The straw is removed from the nitrogen container, placed into a water bath and gently agitated until all ice is melted.
-          The straw is then emptied into a prepared small Petri dish, containing a mixed 10% glycerol and sucrosis in holding medium and kept for 5 minutes.
-          Then the embryo is transferred into another small Petri dish, with 5% glycerol and sucrosis solution in holding medium.
-          Finally the embryos are placed in normal holding medium for 10 minutes before evaluation.
-          The thawed embryos are transferred.

7.   Hints for Donor Preparation



Organisation and Preparation of an Embryo Transfer (E.T.) Programme

The success and the cost involved in embryo transfer depend upon the conditions under which the E.T. is carried out.
1)                 Only animals with undisturbed general well being and healthy and fit are suitable donors.
2)                 Animals with hoof problems, mastitis, uterus infections, metabolic disturbances or any other disease are not suitable as donors.
3)                 Donors with calving problems, premature births, abortions, retention of placenta or any other disturbances must be treated before preparation for embryo transfer.
4)                 Problem animals (repeat breeders) must be announced as such and treated according to their pre-history because sterile cows do not produce viable embryos.
5)                 The superovulation should be started after the cow has shown at least 2 visible heats (oestrus) after calving. Cows with a history of veterinary treatment, or after disease should show at least one visible heat before
programming for superovulation.
6)                 To recognise functional disturbances early, the first heat and the subsequent heat should be confirmed with the Milk Progesterone test, i.e. one sample (after milking) on the day of heat and a second sample one week after heat are taken for testing. Further samples are taken and tested during the superovulation program.

Fig 7. : eProCheck®  for estrus checking


7)                 Embryo Transfer becomes problematic in herds where cows are stabled on steel grids and fixed with chains. This changes the spontaneous behaviour of the cow into restrained behaviour. Under these conditions not all animals adapt (behave) in the same way and therefore cows which undergo treatment for Embryo Transfer should be moved to a place where there is minimum stress.
8)                 The occurrence of an error or disorder whilst preparing the program should be announced as quickly as possible to the responsible person so that the error or the disorder can be compensated.

Very common and accidental errors or disorders are:
- Cow suddenly gets mastitis, goes lame or goes off feed (digestive disorders)
- Incorrect substance injected
- Error in injection schedule
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