Puppy development - the early days !

If you're at all like me, it's not enough to know that she's pregnant and then then sit back and wait for the delivery. I want to know what's happening throughout the whole process, especially cause the wonders of   embryo/foetal development amaze me - it's a true miracle :)

The development is split into 3 stages : Pre-attachment, Embryonic and Foetal.

1. Pre-attachment
After fertilisation there are two nuclei in each egg, one from each parent carrying half of their chromosomes. When these fuse, the puppy is created carrying the full complement of 78 chromosomes  (as opposed to humans with 46, sheep 54, horse 64 and a Kingfisher at 132 !). 8 to 9 days after fertilisation they travel down into the uterus.

2. Embryonic stage
Placentas start to develop about 17 days after fertilisation and at around day 20 the embryos (less than a half an inch long) begin to attach themselves to the walls of the uterus. The central nervous system is developed at this stage. Over the next week the brain and spinal cord develop along with the rest of the organs, limbs, head, eyes and face.

3. Foetal stage
At around 30 days after fertilisation, male and female characteristics show. Over the next few weeks rapid development of the foetus occurs and from day 57 (after fertilisation), live full term puppies can be born. The normal duration of pregnancy is 60 days (+/- 1 day) from fertilisation (or about 63 days from ovulation).

Pregnancy diagnosis

It's been 2 weeks now since Jess was mated, and another 2 week until we're off to the vet for an ultrasound, so another 2 weeks to wait to find out if she is definitely pregnant or not !

Unlike humans, there is no immediate test that can be done to check pregnancy in dogs.  The options available are :

1. Wait until visible signs appear - these are not going to be visible until 35-40 days into the pregnancy though and can be :
- darkening colour and development of the teats
- enlarged mammary glands
- opaque or white mucus vaginal discharge
- morning sickness (as well as refusing food till midday - with a Labrador ? yeah right :)
- abdominal enlargement - this is not likely until day 40-50. When the uterine horns become very full with the developing foetuses they fold back upon themselves and drop lower into the abdomen.  This may not happen if the litter is very small

2. Abdominal palpitation. This involves the vet trying to feel the tiny marble-sized foetuses in the uterus, strung out like a bead necklace. This is possible at around 20-30 days post mating but not on all bitches and it depends on the shape of the bitch

3. Measuring levels of hormone relaxin. This hormone is produced by the placenta and begins to appear in the blood stream at the time when the fertilised eggs become implanted in the womb (usually between 17 and 19 days post-ovulation). This test is normally carried out around 30 days after mating.

4. Ultrasound. Using the same ultrasound imaging equipment as used for women, foetal heartbeats can be seen from days 20-25 of pregnancy.  The accuracy of the results though depend very much on the equipment being used and the skill and expertise of the operator. It is more useful than other tests as it can help to indicate the number and size of the puppies. Pregnancy diagnosis is said to be 94% correct through the use of ultrasound (not sure of the reliability or sample size of that statistic though !), but assessing the number of pups in the litter is not so accurate, especially when there are more than 6 foetuses.

So, it's ultrasound for us (at the preference of the vet) and in 2 weeks time I will be able to post the results :)

Second mating

Yesterday I went back down to Brigburn kennels. Jess did less complaining this time and was far more relaxed once Reed was in. They tied again for 20 min so hopefully as I sit here typing, Jess has a number of fertilised eggs in her uterus :D. Now comes the next wait - it's about 3 weeks before any sort of pregnancy diagnosis can be made. I'll cover the various options in a later post.

Cost : Petrol £35

First mating

Well after what seemed an endless parade of testing, the day finally arrived for Jess to mate with Reed.  Knowing that Jess has never been very keen for anyone paying her too much attention round her back end, I was a little concerned that she might make it very difficult for Reed to do the deed !  We introduced the two together and Reed wasted no time getting down to business :) However, Jess was not impressed - seemed she expected some wining and dining from her men first !  We let them wander round and attempt things on their own for a while and after about 10 min Jess started to warm to the idea so Lynda steadied/assisted things from the back while I kept Jess from wriggling too much from the front.  There were a few yelps and squeals from Jess but once Reed was in and the tie started, she seemed perfectly calm about it all. They tied for about 20 minutes and Jess was happy to stay still for the whole time (although I sat with her holding her collar).

It was great to experience, to see mother nature at work with the dogs natural instincts guiding them through the process.

I'm going to go back again on Wednesday for another mating. I'm wondering whether Jess will be more receptive towards the process this time, or whether once she realises where she is, refuse to get out of the car !

Cost : Petrol (£35), Stud dog fee (£400)

Day 11 - Second progesterone test

Second progesterone test was done on Friday (Day 11) - used my own vet this time, bit more expensive for the consultation but they were able to spin the serum out from the blood for me so Croft would be able to obtain a better reading.  Sent sample off Saturday next day delivery and had the call from Croft at 11am on Saturday.

Reading this time was 7.1 nmol/L which was exactly what I was hoping for. This indicates that the LH surge has taken place, and has elevated the progesterone up from it's Monday level of 1.6 nmol/L (and also indicates that ovulation not yet occurred).  The blood sample was done at 10am on Friday, so LH surge probably occurred late Thursday/early Friday.  From this, the date of ovulation can be determined as being Saturday or Sunday (remembering this is not an exact science down to the nearest hour but can probably pinpoint events within 24 hours if tested at the right times).

The recommendation for the best time for mating is 1 and 3 or 2 and 4 days post-ovulation. So assuming ovulation is taking place on Saturday or Sunday, I'm going for a mating on Monday and Wednesday.  The eggs take approximately 2 days to become fertile after being released (at ovulation) and then remain fertile for another 2-3 days. Fresh semen can remain viable in the reproductive tract for 5-6 days so the combination of the mating days above will provide maximum contact between the fertile eggs and healthy sperm.

So, science and testing takes a rest now, and Mother Nature is being called in :)  Jess has no idea what's in store for her !  Time to refresh myself a bit more about the mating process but luckily I'll have Lynda there with her wealth of experience to guide us through the process !

Cost  : £30 for the test, £30 for my vet to collect the blood sample and £5 postage (£65).

Laboklin results back

Got results back from Laboklin today - great news. Jess tested clear for both EIC and CNM - Yay !

So in total, Jess' health checks were:
Hips : 0/0
Elbows : 1/0
prcd-PRA : Clear
EIC : Clear
CNM : Clear

In combination with Reed :
Hips : Jess 0/0, Reed 4/4
Elbows : Jess 1/0, Reed 0/0
prcd-PRA : Jess Clear, Reed Clear => All puppies clear by parentage
EIC : Jess Clear, Reed Carrier => Approx 50% puppies clear, 50% carriers
CNM : Jess Clear, Reed Clear => All puppies clear by parentage

So DNA testing confirms that Jess and Reeds puppies will never be affected with prcd, EIC or CNM - how comforting :)  There's probably not many litters out there that can claim that (prcd and CNM probably, but not all three).

Of course, Jess still has to get pregnant !  I'm getting a little ahead of myself :D

Results back from first progesterone test

Wow, hats off to Croft. I posted the sample at 3pm yesterday and had a call from Crofts with the result today at 12pm :)  The results were to be expected, progesterone levels at 1.6 nmol/L (day 7), indicating that the LH surge is not yet happened (on average day 10).

To avoid the cost of a third test, I really want the next test to at least show that the LH surge has happened (progesterone levels will be > 6.0) mol/L) so then I will know when ovulation will/has occurred and I can plan the visits down to see Reed :) Friday this week will be day 11 which "should" be in between the days of the LH surge and ovulation. Thankfully Croft is open on the weekend so a next day delivery on the Friday will see me with the results around Sat lunchtime (day 12).

Day 7 - First progesterone test

Jess and I are off to the vets today to take the first blood sample for Croft. Will post the sample down via next day delivery and find out tomorrow the results of the progesterone testing. I expect it to be low (< 2 nmol/l) indicating that the LH surge has not yet happened. They will advise when I should do next test - probably Thursday or Friday (day 10/11) - at which point levels should indicate the LH surge has happened.

Cost  : £30 for the test, £20 for my vet to collect the blood sample and £5 postage (£55).

Progesterone testing

In my last post I talked about the hormones oestrogen and progesterone and their affect/involvement in controlling the oestrus cycle.  About 2 days prior to ovulation a surge in the secretion of LH causes the ovarian cells to switch from secreting oestrogen to progesterone. Prior to this LH surge, progesterone levels are very low . On the same day of the LH surge, progesterone levels will start to rise, continuing to do so through to ovulation and beyond. Once the bitch has ovulated, progesterone levels can vary greatly so the important test is knowing exactly when the progesterone has passed the ovulation mark. Once it's passed, you can't accurately predict how long ago ovulation took place.

Knowing the day of ovulation allows you to maximise contact time between viable semen (which can remain viable in the bitch's uterus for 3-5 days) and fertile eggs.  Once ovulated, the eggs take approximately 48 hours to become fertile. They will then remain fertile for approximately another 48 hours. The normal recommendation for mating is therefore days 1 and 3 or 2 and 4 post-ovulation.

So, on average, the LH surge occurs on day 10 (where day 1 is the first day where bleeding is detected in the bitch), ovulation 2 days later on day 12 and mating on day 14 and 16 (2 and 4 days post ovulation).

As mentioned earlier, prior to ovulation, progesterone levels as very low (less then 2 nmol/l). When the levels of LH surge, progesterone levels will quickly rise to above 6 nmol/l. At the time of ovulation, levels will be around 15-20 nmol/l. By detecting the change in progesterone levels around the LH peak, the date of ovulation can be accurately identified. While the LH surge is on average around day 10, it can be as early as day 5 or as late as day 25, which is why it's usually advised to start testing about day 5-7. Depending on the results from the first test, the lab will advise when next to test (usually about 3 days later).

There are "off the shelf" or "in house" kits that many vets stock (like pre-mate) that test for progesterone levels. It's important to realise that while they can give you a quick result (within the hour), they are qualitative tests, and not quantitative and are unable to provide you with accurate results. These simple ELISA based tests use colour changes to predict the levels of progesterone in the blood, with a stronger colour changes indicating higher levels. Lots of people have used them and swear by them, but there are just as many stories about them not working, due to a number of contributing factors (age of test, temperature, subjective colour analysis etc.).

Personally, I would only recommend the laboratory quantitative tests - I don't see the point in spending roughly the same amount of money (about £30) for a test that is not as accurate.  This is the place I am going to use Croft Reproduction. The have staff working 24x7 and will phone you on the same day that the blood sample is received. Can't ask for better service than that :) There are some other labs that will do the same test like Cambridge Specialist Labs but they are only open Mon-Fri business hours which is frustrating if you need results over the weekend !

The cost with Croft Reproduction : £30 for the test, £20 for my vet to collect the blood sample and £5 postage (£55).

I'm hoping that 2 tests will be enough, I really must learn how to take the blood myself - certainly doesn't look difficult and would save me £20 each time ! ;)

Season and Ovulation

These two things are very important when it comes to knowing the best time to mate your bitch.  Ovulation is identified by the levels of progesterone in the blood (there are other ways as well but this is the most reliable). To understand how progesterone testing provides you the answers, I'll first explain a little about the reproductive cycle and the hormones involved.

The normal reproductive (oestrous) cycle of a bitch is comprised of 4 stages (although only the first two are when the bitch is "in heat")

1. Pro-oestrus (average 9 days but can range from 2 to 27). This is the beginning of heat. The vulva is swollen and there is a blood-stained discharge. Although the bitch is (very:) attractive to dogs, she will not allow them to mate her
2. Oestrus (average 9 days but can range from 3 to 21 days). This stage is when the bitch will accept the male. The vulva is very enlarged. The discharge is straw-coloured rather than blood-stained. Ovulation occurs spontaneously usually about 2 days after the start of oestrus
3. Diestrus (average 2 months). Approximately 6 days after ovulation the diestrus stage starts. This stage ends just prior to whelping in the pregnant bitch or approximately 2 months after ovulation in the non-pregnant bitch.
4. Anestrus (average 4 months). Duration of anestrus is quite variable among bitches and may be governed by both genetic and environmental variables.

The physiology of the oestrus cycle can be summarised by

- The hypothalamus (part of the brain) causes the production of "follicle stimulating hormone" (FSH)
- FSH controls the development of the ovarian follicles, which in turn secretes the sex hormone oestrogen
- Low levels of oestrogen simulate further FSH to be released and therefore rising levels of oestrogen
- The above process continues until the follicles are mature and about to rupture at which points the high levels of oestrogen start to inhibit FSH but also trigger the release of "luteinizing hormone" (LH)
- It is the release of LH that causes ovulation and initiates the development of the ruptured follicle into a solid body, the corpus luteum
- The corpora lutea secrete progesterone
- High levels of progesterone inhibits the production of FSH and LH
- It is the balance of the two sex hormones oestrogen and progesterone that ultimately controls the oestrus cycle

In my next post I will go in to more detail about the level of progesterone at the various stages and what it tells you about ovulation and the ideal time to mate

Coat colour genetics

Labrador Retrievers come in three distinct colours: black, yellow and chocolate. The colour of a dog depends on the presence of pigments in skin and hair. The pigments occur as two distinct types. The first type is essentially yellow. The second one is dark, and varies from black to brown. The type of pigment that a Labrador produces depends on its genetic make-up and is the result of an interaction between two different genes, which are called the E and the B gene.

A dog's genome consists of two sets of genes, one from each of the parents. So for each gene, if different forms exist, a dog can have two forms of the gene.

Different forms of the E and the B gene exist. The difference in the genes is distinguished by uppercase and lowercase letters. So the E gene exists as E and e, while the B gene exists as B and b. Therefore, depending on the parents, a dog genome may be EEBB, EeBb, eeBb or any other combination.

The form that is written in an uppercase letter is called the dominant form. This means that, if a dog has both forms of a particular gene, it is the form that is written in capitals that has an effect on the dog — it is expressed. If there is no dominant gene the recessive (lower case) one will be expressed. For example, for a dog with eeBb, the e and the B forms will be expressed.

Black dogs can have 4 possible genomes
- BBEE : pure (dominant) black
- BbEE : black carries chocolate
- BBEe : black carries yellow
- BbEe : black carries yellow and chocolate

Yellow dogs can have 3 possible genomes

- BBee : yellow carries black

- bbee : yellow carries chocolate

- Bbee : yellow carries black and chocolate

Chocolate dogs can have just 2 possible genomes

- bbEE : pure chocolate

- bbEe : chocolate carries yellow

A great chart showing the possible combinations and outcomes can be found here

http://www.vetdnacenter.com/files/coat-color-predictor.pdf

Of course these just show the probability of the outcome. I don't know which type Jess is (bbEE or bbEe) must most chocolate dogs are bbEE as matings between yellow and chocolate is not common (partly in reason as it can produce yellow pups liver noses). Regardless of Jess' genome, Reed is a black carrying chocolate (BbEE) so the probable outcome is the same (as can be seen from the chart above) : 50% chocolate pups and 50% black pups. Of course you may get 100% black or 100% chocolate but somewhere in between is a more likely outcome.

In case this has only just whet you're appetite :) a far more detailed look at colour coat genetics can be found here

http://www.labbies.com/genetics2.htm

Choosing a suitable stud - Part 3

A couple of other things that I forgot to mention earlier.  Helping me make my mind up about show vs. working lines was some advice given that a lot of people looking for pups to buy as pets (and nothing more) would prefer  to see a mixture of show and working lines rather than a pup bred purely from working lines, as they knew they wouldn't have the time or energy to devote to a very active dog. Some good advice I thought, although clearly these puppy buyers are more educated than I was when I looked for a pup - I was certainly not aware of differences such as size, shape and energy levels between show and working lines when I was looking !

The other suggestion was to mate Jess with a black carrying chocolate (rather than dominant black), as some of the pups then will be chocolate, which at the moment, are easier to sell than black pups (I'll cover coat colour genetics in a later post).

I figured that the more people I could attract to Jess' pups then the better chance I will have of a/ finding them all a good home and b/ making sure I was happy with the quality of life the new owners were going to provide for them.

EIC and CNM

EIC stands for Exercise Induced Collapse. It is an recessive inherited condition like prcd-PRA (so the same possible outcomes of affected/carriers and clears exists as mentioned in my earlier post). Affected dogs can endure mild to moderate exercise but after 5 to 20 minutes of heavy exercise with extreme excitement, the dog shows weakness and then collapse. Severely affected dogs may collapse whenever they are exercised to this extend – other dogs only exhibit collapse episodes sporadically.

Signs of EIC are not typically seen until the dog begin intense training. First symptoms are usually noted between 5 months and 3 years of age. However, it is confirmed that some affected dogs did not have collapse episodes until as late as age 10.

You may think that having a dog collapse is not that bad, certainly not as bad as going blind, but a youtube video showing a dog having an EIC episode was so bad that I could not watch more than the first minute - I would not wish that on any dog.  And, it is possible that an EIC collapse can lead to the death of the dog.

CNM stands for Centronuclear Myopathy. It also is a recessive inherited condition. Affected dogs show symptoms including hypotonia, generalized muscle weakness, abnormal postures, stiff hopping gait, exercise intolerance and increased collapse when exposed to cold.

So, because I believe its the most responsible thing to do, especially with Reed as a known carrier of EIC, I felt I had to test for both diseases. If Jess comes back clear for EIC then the pups will be 50% clear, 50% carriers (but none of them will ever suffer from this disease). If Jess comes back clear for CNM then the pups will all be 100% clear (as Reed is clear).  This, combined with the prcd-PRA test will mean (assuming jess is clear for both of these - or even if she is a carrier of CNM) that all of the pups will never suffer from prcd, EIC or CNM - certainly I think that is a good thing :)  And very comforting for the new puppy owners.

I used Laboklin for both of these tests

http://www.laboklin.co.uk

Cost : Blood sample at vet £20, Postage £7, EIC test £67, CNM test £50 - total £144

More DNA testing

Lynda, much like myself is a strong advocate for testing against disease and using everything available at our disposal to ensure the best possible outcome for any pups.  It's fairly new at the moment and a lot of people are not prepared to add to the list of tests already done (I won't go into all the arguments for and against here else I will never get this blog up to date !). My concern is that for most of the people that refuse to take tests that are available is that they are scared of the outcome.  That and people's reaction to using a dog identified as a "carrier". I think that some people are wary of using a dog that's been identified as a carrier, when all that really means is that you make sure you breed with a dog that is clear.  While they don't like the idea of a carrier, they seem to be more comfortable with not doing the test at all ? That is crazy to me - to be more comfortable with the unknown (and perhaps mating with an affected dog) rather than knowing the dog is a carrier (who will never be affected).

Anyway, I feel myself starting to rant so I will stop. There are lots of discussions/opinions out there on just how far to take the whole testing process. For me, I believe I needed to do all I could for the best possible outcome for both the puppies and their new owners !  Of course you could also argue that pedigree breeding (and historically lot's of very close line breeding or inbreeding) have produced all these results in the first place ;)

I absolutely loved Reed and felt like this was the dog I wanted to use so, since Reed is a carrier of EIC, I had to get Jess tested as well (at the moment, I have not had the results back - if she comes back a carrier then it's back to the drawing board !). It's typically a problem in the show lines though so Jess should be fine. While I was sending results off anyway, I decided to do the test for CNM as well. Reed has tested clear but CNM is a problem more with the working lines so figured I should put that to rest as well. I will explain these two tests in following posts.

Choosing a suitable stud - Part 2

The next step was speaking to some people who knew more than me (ie something :)  I spoke to the VP of the Labrador Club of Scotland first off as well as an experienced breeder from the Labrador Forums who kindly spent lots of time making up sample pedigrees and fielding lots of questions (thanks Sue !!) and a few other people who had spent a long time in the "industry".

They all arrived at the same suggestion, that mating Jess with a show line could help to produce calmer, more rounded puppies (more suitable for day to day life). For those who don't know, Jess is extremely wired !  She could happily tear through fields for hours only to start the whole thing over again. Super high amounts of energy, which for some people, would be too much (sometimes is for me also :) I initially thought I would be breaking some special "code" amongst breeders by crossing working lines with show lines but it seemed not the case, in fact everyone agreed it was the best way for me to move forward.

So all that work, whittling down all my working stud dogs was tossed out the window and I felt like I was back at square 1 :(  There were a lot of show line studs that I just didn't like the look of though, so that reduced the numbers (perhaps not the most sensible grounds for elimination ;) I was keen also to have some element of working ability in the dog (didn't want to lose all of Jess' fire and love of hard work). Two people highly recommended the same kennel/dog - Charway Water Reed of Brigburn Kennels.

As soon as I saw Reed on the Brigburn website, saw him diving in to the water and hearing how Lynda (Brigburn owner) had a lot of experience in both the show and working lines, I knew I had found my man (so to speak ;)  A chat with Lynda who told me how Reed loved going out on hunts (and was proving very capable) further confirmed this.  I also appreciated Lynda's no-nonsense attitude and considerable experience and I at once felt comfortable working with her.  So, a trip down to see Reed was made (cost £35 petrol) - but we got a free coffee :D

Here is the magnificent Reed in all his glory

http://www.brigburn.com/html/reed.html

Choosing a suitable stud - Part 1

I'm still playing some catch up here, hopefully after this weekend I'll be up to date :)

So once the hips, elbows and eye certificate had passed ok it was time to start considering which stud dog to use. Initially I had no idea there were so many !  I had started off by just looking at the champdogs site and their list of stud dogs

http://www.champdogs.co.uk/guided/all_labrador-retriever_stud-dogs.html

I at least knew I wanted a black stud as we want to keep a black puppy so that was the colour out of the way - the choices were still overwhelming. I wanted hip scores as low as possible, certainly under 10, and because of Jess' elbow scoring, had to have a stud with elbows of 0/0.  DNA testing for pra was also mandatory.  All the dogs fell within these guidelines though !

I started to look back through Jess' pedigree as I knew a popular breeding technique was "linebreeding" - not tobe confused with "crossbreeding". Another category that exists is "outcrossing". They are all described well here

http://www.labradorforums.co.uk/ftopict-12936-outcrossing.html

Jess' 5 generation pedigree had mostly dogs from field trial backgrounds (FtCh - Field Trial Champion, FTW - Field Trial Winner). There were however also some (mostly from her mothers side) from the show lines (ShCh - Show Champion, CH Show Champion that has gained it's show dog working certificate). I was keen to persue field work with both Jess and one of her pups so initially I narrowed my search down to working/field trial dogs with strong FtCh's in their pedigree.  I also preferred the more athletic, leaner look of a working dog compared with a show dog (in general). So my criteria - hips (5/5 or lower), elbows 0/0, pra carrier/clear (preferably clear) and field trial pedigree.....or so I thought :)

Optigen results

Results came in the post today for Jess' prcd-PRA test. Results show she is Normal/Clear (YAY !), meaning that she carries no copy of the mutant gene.  Neither does the stud dog so that means all puppies will be normal/clear for prcd-PRA by parentage. We're breeding a whole generation that is guaranteed not to ever develop prcd-PRA :)

The countdown has started !

YAY :) This morning Jess officially started her season (or in technical terms has just started the first of 4 stages of her reproductive cycle - Proestrus)   That means approximately 12 days until ovulation (with day1 being today), so optimum mating is on day 14 and 16 (ova takes 48 to 72 hours to mature after ovulation and they remain fertile for another 2 to 3 days). We will be taking blood samples and quantitatively testing for progesterone levels which will help us to pinpoint the time of ovulation (since ovulation can occur anywhere between day 7 and day 21, but 11-13 is the norm).

Photos

I've added a new page "Photos" which will have just that, some pics of Jess